REVIEW ARTICLE
AT-rich region and repeated sequences – the essential elements
of replication origins of bacterial replicons
Magdalena Rajewska, Katarzyna Wegrzyn & Igor Konieczny
Department of Molecular and Cellular Biology, Intercollegiate Faculty of Biotechnology, University of Gdansk, Gdansk, Poland
Correspondence: Igor Konieczny,
Department of Molecular and Cellular
Biology, Intercollegiate Faculty of
Biotechnology, University of Gdansk, Kladki
24, 80-822 Gdansk, Poland. Tel.: +48
585 236 365; fax: +48 585 236 427;
e-mail: [email protected]
Received 15 March 2011; accepted 7 July
2011. Final version published online 25
August 2011.
DOI: 10.1111/j.1574-6976.2011.00300.x
Editor: Grzegorz Wegrzyn
MICROBIOLOGY REVIEWS
Keywords
replication origin; 13-mers; DUE; Rep; DnaA.
Abstract
Repeated sequences are commonly present in the sites for DNA replication initiation in bacterial, archaeal, and eukaryotic replicons. Those motifs are usually
the binding places for replication initiation proteins or replication regulatory
factors. In prokaryotic replication origins, the most abundant repeated
sequences are DnaA boxes which are the binding sites for chromosomal replication initiation protein DnaA, iterons which bind plasmid or phage DNA
replication initiators, defined motifs for site-specific DNA methylation, and
13-nucleotide-long motifs of a not too well-characterized function, which are
present within a specific region of replication origin containing higher than
average content of adenine and thymine residues. In this review, we specify
methods allowing identification of a replication origin, basing on the localization of an AT-rich region and the arrangement of the origin’s structural
elements. We describe the regularity of the position and structure of the
AT-rich regions in bacterial chromosomes and plasmids. The importance of
13-nucleotide-long repeats present at the AT-rich region, as well as other
motifs overlapping them, was pointed out to be essential for DNA replication
initiation including origin opening, helicase loading and replication complex
assembly. We also summarize the role of AT-rich region repeated sequences
for DNA replication regulation.
Introduction
Although it has been almost 50 years since the replicon
model was originally proposed by Jacob and Brenner
(1963), our understanding of the concept of the replication origin is still evolving. It is generally accepted that
the replication origin is considered to be a site within the
DNA particle where the replication is initiated; however,
the sequence specificity and structural requirements of
this important genetic element are not fully elucidated
and vary depending on the analyzed replicon. Although
the bacterial origin sequences are usually well defined, the
eukaryotic and in particular higher eukaryotic replication
initiation sites are not characterized so well. The identification of the replication origin is still difficult and
requires not only the analysis of the DNA sequences
in silico but also application of advanced experimental
approaches.
The replication origins of the bacterial chromosomes,
bacteriophages and plasmids, as well as certain eukaryotic
FEMS Microbiol Rev 36 (2012) 408–434
replicons, including the DNA viruses and Saccharomyces
cerevisiae, possess characteristic functional elements,
including specific binding sites for the appropriate replication initiation protein (Rep), binding sites for accessory
replication proteins, and regions with high content of adenine and thymine residues (AT-rich) (Bell & Dutta, 2002;
Konieczny, 2003; Breier et al., 2004; Mackiewicz et al.,
2004; Ozaki et al., 2006; Chew et al., 2007; Shaheen et al.,
2009). The distribution of DnaA-boxes which are the
binding sites for the bacterial replication initiation
protein DnaA and the dnaA gene location could be
applied for the identification of the bacterial chromosomal
replication origins (oriC) (Mackiewicz et al., 2004). Moreover, the asymmetry in nucleotide composition measured
as the normalized difference in the content of complementary nucleotides is considered to be the method of
identification of putative oriC in bacterial chromosomes
(Freeman et al., 1998; Grigoriev, 1998; McLean et al.,
1998; Salzberg et al., 1998; Mackiewicz et al., 1999a, b;
Rocha et al., 1999). It has been demonstrated that the
ª 2011 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
AT-rich region and the repeated sequences in DNA replication
DNA asymmetry is the most universal method but to
achieve a better prediction, it is proposed to apply it
together with the analysis of the distribution of DnaA
boxes and dnaA gene location (Mackiewicz et al., 2004).
Unfortunately, these characteristic features, i.e. a cluster
of DnaA boxes, a dnaA gene and a switch in the asymmetry, are neither typical of all bacterial chromosomes
(Mackiewicz et al., 2004) nor of other replicons such as
plasmids and bacteriophages. By contrast, the AT-rich
part of a replication origin is the most universally conserved structural element identified in both prokaryotic
and eukaryotic replicons (Bell & Dutta, 2002; Konieczny,
2003; Breier et al., 2004; Mackiewicz et al., 2004). This
distinct region forms the DNA unwinding element (DUE)
(Kowalski & Eddy, 1989) where during the process of replication initiation, the initial destabilization (opening) of
the DNA double helix takes place. The opening creates a
single-stranded DNA (ssDNA) which is indispensable for
the replication initiation of a double-stranded (dsDNA)
replicon. The AT-rich region is exactly the site where a
replication complex is formed and where the DNA synthesis is initiated. Surprisingly, our knowledge about the
AT-rich region is limited and very often the conventional
perception is that the only important feature of this part
of the replication origin is its higher than average number
of adenine and thymine residues. Recent data revealed,
however, that the sequence of the AT-rich region is of
great importance.
Localization of the AT-rich regions
within replication origin
The replication process starts in the AT-rich sites within
one, two or multiple origins, depending on the organism.
The vicinity of these specific regions is characteristic of
the particular replicon; however, there are several conserved motifs surrounding the AT-rich sequences that can
be recognized within different replication origins. To
determine the location of the individual regions at the
origin, identification of the replication origin itself is
required in the first place.
Methods for the identification of replication
origin
The identification of a replication origin requires application of a wide range of techniques from the prediction of
the origin sequence in silico to its in vivo and in vitro
confirmation. The simplest theoretical indicators of the
replication origin’s position in bacterial chromosomes
take the advantage of the differences in the GC and the
AT content of leading and lagging strands. The observed
deviation in nucleotides composition between two strands
FEMS Microbiol Rev 36 (2012) 408–434
409
is more likely the effect of different mutation rates connected with the architectural asymmetry at the replication
forks and the higher accuracy of leading strand replication (Mrazek & Karlin, 1998; Frank & Lobry, 2000; Tillier
& Collins, 2000). The relative nucleotides skews (e.g. CG
skew = (C
G)/(C + G)), which are normalized differences in the content of complementary nucleotides,
change the sign crossing replication origin (Lobry, 1996a,
b; Freeman et al., 1998; Grigoriev, 1998; McLean et al.,
1998; Salzberg et al., 1998). The observed asymmetry in
the nucleotides composition is used, for example, in the
Oriloc (Frank & Lobry, 2000) and the CG-software (Roten
et al., 2002) programs for origin prediction. These computational methods designed for bacterial origin identification can also be applied to predict the sites where the
replication starts in archaea, despite the fact that the
archaeal replication apparatus appears to be more closely
related to that in eukaryotes (Grigoriev, 1998; Lopez
et al., 1999; Myllykallio et al., 2000; Zhang & Zhang,
2002, 2005). However, the analysis of chromosome asymmetry only gives the approximate origin position within
the genome sequence, indicating the region with a higher
than average AT content. For a more efficient determination of the site where the replication starts, other origin
features, such as the dnaA gene and the position of
the DnaA-box motifs, should be taken into account
(Mackiewicz et al., 2004). Considering the location of the
gene for bacterial initiation replication protein DnaA is
even more justified because the dnaA was found in
almost all bacterial chromosomes (Mackiewicz et al.,
2004) and the origin for replication is usually located
upstream or downstream of the dnaA gene (Messer,
2002). Moreover, using the position of the sequences recognized by this protein, which were found within the replication origin in almost all analyzed prokaryotic genomes
(Mackiewicz et al., 2004), is equally justified. What is
interesting, as the dnaA gene in bacterial chromosomes is
located near the replication start point, similarly the
archaeal genes for replication initiation proteins Orc1/
Cdc6 are also found close to the origin and their position
could be used for origin prediction (Norais et al., 2007)
The parameters like the dnaA and DnaA-boxes’ position
or the location of indicator genes (hemE, gidA, dnaN,
hemB, repC, etc.) and dif sequence are applied in DORIC
database (Gao & Zhang, 2007) and ORI-FINDER web server
(Gao & Zhang, 2008). The DORIC database contains over a
thousand deposited sequences (http://tubic.tju.edu.cn/
doric/). However, it should be taken into account that
sometimes applying different parameters in the in silico
studies can generate different results. The analysis, presented by Mackiewicz and coauthors, of 112 complete
bacterial genome sequences, based on three parameters,
in case of 67 chromosomes indicates the same region as
ª 2011 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
410
replication origin; however, in case of 17 genomes each
parameter indicates a disparate region (Mackiewicz et al.,
2004). As the available programs usually base on the
Escherichia coli oriC features and in some bacteria, there
can occur substantial differences in DnaA-boxes’ sequences
like in Helicobacter pylori (Zawilak et al., 2003) and
Borrelia burgdorferi (Picardeau et al., 1999) or variations
in the dnaA gene position (Mushegian & Koonin, 1996;
Richter et al., 1998), therefore the prediction of the replication origin location cannot be approved indiscriminately
without experimental data. The conduction of experiments
is extremely important in case of organisms whose genomes contain more than one region where the replication
starts, as different methods could indicate various regions
as replication origins (Dijkwel & Hamlin, 1995; Pelizon
et al., 1996; Berquist & DasSarma, 2003; Lundgren et al.,
2004; Robinson et al., 2004; Zhang & Zhang, 2005; Coker
et al., 2009).
The first experiments leading to the determination of
bacterial origin relied on construction of minichromosomes and investigation of their ability to replicate in
bacterial cells (Meijer et al., 1979; Messer et al., 1979; Sugimoto et al., 1979; Oka et al., 1980). Then the in vivo
analysis was extended to in vitro experiments to confirm
the site where the replication starts using techniques such
as two-dimensional gel electrophoresis (Yee & Smith,
1990; Suhan et al., 1994) or electron microscopy (Inselburg, 1974; Tomizawa et al., 1974; Krause et al., 1997),
which allowed to observe the replication intermediates.
These methods of origin identification were also successfully applied in case of archaeal and eukaryotic ARS
(autonomously replicating sequence) regions (Stinchcomb
et al., 1979; Hamlin & Dijkwel, 1995; DePamphilis, 1999;
Matsunaga et al., 2001; Lundgren et al., 2004; Robinson
et al., 2004). To determine where the replication proteins
bind within the origin specific motifs, the EMSA (Electro
Mobility Shift Assay) (Margulies & Kaguni, 1996; Weigel
et al., 1997; Zawilak et al., 2003; Ozaki et al., 2006; Sibley
et al., 2006), footprinting (Hwang & Kornberg, 1992a;
Margulies & Kaguni, 1996; Speck and Messer, 2001; Shaheen et al., 2009) and SPR (surface plasmon resonance)
analysis (Messer et al., 2001; Speck and Messer, 2001;
Zawilak et al., 2003) were performed. The use of these
methods enabled not only to determine whether or not
the protein binds within the replication origin, but also
to identify the sequence recognized by the protein and to
calculate the stoichiometry and kinetics of the nucleoprotein complex formation. The in vitro helix opening assays
with KMnO4 probing and P1, S1 or mung bean nucleases
were also performed to find the site, where helix melting
occurs and where the replication complex is exactly
formed (Gille & Messer, 1991; Mukhopadhyay et al.,
1993; Krause et al., 1997; Speck and Messer, 2001; Ozaki
ª 2011 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
M. Rajewska et al.
et al., 2006). Analogous biochemical analyses were applied
to determine the plasmids’ replication origins, for
instance the ColEI (Tomizawa et al., 1977), P1 (Abeles,
1986; Abeles et al., 1990; Wickner et al., 1990; Papp et al.,
1993; Park et al., 1998, 2001; Park & Chattoraj, 2001), F
(Eichenlaub et al., 1977; Zzaman et al., 2004), R6K (Kunnimalaiyaan et al., 2004), RK2 (Konieczny et al., 1997;
Doran et al., 1998) and other (Danbara et al., 1980; Diaz
& Staudenbauer, 1982; Kuzminov et al., 1997; Diaz-Lopez
et al., 2003; Schvartzman et al. 2010). For identification
of the archaeal and eukaryotic origins, where the recognition of the replication start point is much more difficult,
other methods, such as marker frequency analysis utilizing whole-genome DNA microarrays and replication initiation point analyses (Gerbi & Bielinsky, 1997; Bielinsky &
Gerbi, 1998, 1999, 2001; Abdurashidova et al., 2000; Gomez & Antequera, 1999; Romero & Lee, 2008), have been
applied. However, no matter how difficult the mapping
of the multiple replication start points is and how much
they differ from the bacterial origins, the common feature
for all of the origins is the AT richness within the region
where the replication process starts.
Localization of AT-rich region in respect to
other origin-specific motifs
Looking closer into the bacterial and plasmid origins with
reference to the position of the AT-rich region, there is a
clear noticeable regularity (Fig. 1). In the analyzed replicons, the binding sites for the replication initiating proteins
in different origins are located on the same site of the ATrich region. The best characterized binding sequences for
the bacterial replication initiators are DnaA-boxes, the
binding sites for the DnaA protein. Within the E. coli’s
245-bp oriC, there are five asymmetric 9-mer repeats
R1–R5 (Fuller et al., 1984; Matsui et al., 1985), bound with
different affinity by both active ATP-DnaA and inactive
ADP-DnaA proteins (Schaefer & Messer, 1991; Schaper &
Messer, 1995; Margulies & Kaguni, 1996; Weigel et al.,
1997; Blaesing et al., 2000; Hansen et al., 2006). There are
also, discovered recently, three I-sites preferentially bound
by the ATP-DnaA form (McGarry et al., 2004). Based on
the binding affinity, the sites recognized by DnaA were
grouped into strong affinity sites (R1, R2 and R4) and low
affinity sites (R3, R5 and I-sites) (Leonard & Grimwade,
2005). Both types are necessary for proper origin functioning as, for example, increasing the number of the I-sites by
replacing the R5 box with I2 inactivated the extrachromosomal oriC function (Grimwade et al., 2007). The origin
activity also depends on proper helical phasing of the binding sites for DnaA, as changes of one helical turn between
the R3 and R4 DnaA-boxes did not alter the replication
efficiency, whereas the insertion or deletion of a part of one
FEMS Microbiol Rev 36 (2012) 408–434
AT-rich region and the repeated sequences in DNA replication
411
Fig. 1. Organization of selected chromosomal
and plasmid replication origins. AT-rich region
of each origin is depicted as a shaded red box,
repeated sequences within the region are
marked with black arrows. DnaA-box
sequences are marked as yellow triangles;
iterons – orange arrows. The origins are not
drawn to scale. Specific structural
arrangements characterize both chromosomal
and plasmid DNA replication origins. The
AT-rich regions in bacterial chromosomal
origins of replication are usually located on
one side of a cluster of DnaA-box sequences.
In case of the plasmid origins, this rule applies
to the AT-rich region and the iteron sequences
recognized by initiator proteins. DnaA-boxes,
if present at a plasmid origin, are usually
distributed on the opposite to the iterons side
of the AT-rich region or flank the motifs.
helical turn completely destroyed the oriC function (Woelker & Messer, 1993). All of the E. coli DnaA-boxes are
located in the vicinity of the AT-rich region and the distance between the DnaA-box region and the AT-rich part
of the origin is strictly critical for the origin replication
activity. Even a single nucleotide insertion disabled the oriC
function both in vivo and in vitro (Hsu et al., 1994). Four
and seven DnaA-boxes of a similar location with relation to
the AT-rich region were found in Pseudomonas aeruginosa
and Pseudomonas putida, respectively (Yee & Smith, 1990).
Most of these motifs share the E. coli DnaA-box consensus
sequence 5′-TTATNCACA-3′ and two of them in both
Pseudomonas origins have the same orientation and spacing
as the R1 and R4 boxes in E. coli oriC. Unfortunately, neither the pseudomonad origins can be functional in E. coli
cells nor the enteric origin can be active in Pseudomonas
cells (Yee & Smith, 1990). This phenomenon is not
observed in case of Vibrio cholerae chromosome I and II
origins because E. coli cells can be transformed with plasmids containing either oriCI or oriCII, but only when the
Dam methyltransferase is present in cells (Egan & Waldor,
2003; Koch et al., 2010). From the two V. cholerae origins,
oriCI is very similar to the E. coli oriC and shares its 58%
sequence identity (Egan & Waldor, 2003). Within this
FEMS Microbiol Rev 36 (2012) 408–434
307-bp origin, six sequences recognized by DnaA protein
were found, five of them (R1–R5) are common for all
Enterobacteriaceae and the sixth (RV) is characteristic for
the Vibrio genus. However, this unique RV motif is not
essential for the replication function of the minimal origin
region (Saha et al., 2004). All six DnaA-boxes were identified downstream of an AT-rich sequence (Fig. 1). Similarly
as for gammaproteobacteria, in the replication origin of alphaproteobacteria and other bacterial taxons, the sites
bound by chromosomal replication initiator are observed
along the region abounding with adenines and thymines
(Fig. 1). In Caulobacter crescentus’ origin, five DnaA binding sites were found (Marczynski & Shapiro, 1992); however, none of them shared the perfect oriC consensus
sequence (Schaefer & Messer, 1991). One of the identified
DnaA-boxes, differing just in one nucleotide position when
compared with E. coli DnaA-box, was found also in the
origins of other freshwater and marine Caulobacter sp.
(Shaheen et al., 2009). The introduction of a second mismatch into this sequence resulted in an impairment of origin function (Marczynski & Shapiro, 1992). An exact
match to this C. crescentus DnaA binding site was found in
Sinorhizobium meliloti origin, which additionally contains
four other DnaA-boxes (Sibley et al., 2006). As many as
ª 2011 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
412
ten, different from the E. coli DnaA-boxes, were also found
within a 559-bp origin of a thermophile bacterium
Thermotoga maritima. These 12-mer sequences designated
as tma DnaA-boxes, with the consensus 5′-AAACCTAC
CACC-3′ (Lopez et al., 2000), are bound by tmaDnaA
protein which shares 29% identity and 47% similarity with
the E. coli initiator protein homologue (Ozaki et al., 2006).
However, in contrast to the E. coli DnaA protein, the
tmaDnaA binds to the recognized sites with constant affinity. Even more DnaA-boxes than in T. maritima origin
were found in the origin of Gram-positive bacterium
Bacillus subtilis. Three clusters of DnaA-boxes – incA, incB,
and incC – that contain five, four, and six sequences recognized by DnaA protein, respectively, were identified.
Although the number and organization of the B. subtilis
DnaA-boxes are different when compared with those in
E. coli oriC, in an in vitro assay the E.coli DnaA protein
recognized sequences of Bacillus and vice versa, while the
origin regions were incompatible in vivo (Fukuoka et al.,
1990; Messer, 2002). Between the incB and incC clusters
and upstream of the incA region, a dnaA gene was located;
also there were detected 16-mer AT-rich sequences (Moriya
et al., 1992; Krause et al., 1997). However, the analysis of
the open complex formation after KMnO4 probing revealed
that the melting did not occur in these 16-mer repeats but
in a 27-mer AT-cluster downstream of incC DnaA-boxes
group (Moriya et al., 1994; Krause et al., 1997). Although
the position of the opened AT-rich region in Bacillus origin
is not located upstream of the sites bound by DnaA, it
should be emphasized that DnaA-boxes never outflank the
region where the melting of the DNA helix occurs. Even
when looking at the origin of 1.7-Mbp megaplasmid pSymb from Rhizobium meliloti, it is noticeable that the three
DnaA-boxes are located on one side of an AT-rich region,
which is especially similar to that of C. crescentus. Two sites
for DnaA binding possess the exact consensus sequences
and the third contains one mismatch (Margolin & Long,
1993). There are also potential binding sites for the megaplasmid-specific factor inducing the pSym-b replication,
but they have not been identified yet (Margolin & Long,
1993). As the bacterial chromosomal replication origins
contain at least four conserved DnaA-boxes and many
plasmids contain just two such sites and encode their own
additional initiator protein that binds within the origin,
this megaplasmid combines features of both chromosomal
and plasmid origin (Margolin & Long, 1993).
To conclude, the AT-rich regions in bacterial chromosomal origins of replication are usually located at one side
of a cluster of DnaA-box sequences. Such regular position
of those regions, crucial for the replication initiation, is
possibly connected with the function of initiator proteins
and the mechanism of replication initiation (see paragraphs below).
ª 2011 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
M. Rajewska et al.
The organization of the position of the motifs within
plasmid origins is also not random. The presence of iterons that are tandem repeats bound by plasmid initiator
is characteristic of low-copy-number bacterial plasmids.
And as the DnaA-boxes are usually located upstream of
the AT-rich sequences within the plasmid origin, the iterons are found downstream of the region rich in adenines
and thymines (Fig. 1). Some of the best characterized
plasmids with this type of organization of the region
where replication starts are bacteriophage P1 and F plasmid. The P1 origin consists of two DnaA-boxes upstream
of 7-bp AT-rich repeats followed by five 19-mer iterons.
(Abeles et al., 1984, 1990; Abeles, 1986). To the right of
the iterons, there are also three additional sites bound by
the DnaA protein. However, the results obtained by Park
and Chattoraj showed that the origins with DnaA-boxes
situated closer to the origin-unwinding location opened
more efficiently and allowed plasmids to be maintained at
higher copy numbers (Park & Chattoraj, 2001). Within
the left and right clusters of the DnaA-boxes, there is one
perfect consensus sequence and the rest of the sites contain one or two mismatches. It was shown that the presence of one cluster of the DnaA-boxes is sufficient for
providing origin activity, moreover, just one site recognized by DnaA could support P1 replication, but the box
sequence must possess the exact consensus sequence (Abeles et al., 1990). Located downstream of the DnaA-boxes,
19-mer repeats of sequence 5′-GATGTGTGCTGGCGGG
ATA-3′ (Papp et al., 1994) are bound by RepA protein
and this interaction, as well as DnaA binding, is necessary
for proper origin functioning (Abeles, 1986; Wickner
et al., 1990). Similarly, in the F plasmid, binding a plasmid initiator protein RepE and the bacterial DnaA is
required for an efficient plasmid replication initiation
(Hansen & Yarmolinsky, 1986; Kline et al., 1986; Murakami et al., 1987; Masson & Ray, 1988; Ishiai et al., 1994;
Kawasaki et al., 1996). The sites where these proteins
bind, according to the observed rules, are located
upstream (DnaA-boxes) and downstream (iterons) of the
AT-rich region (Fig. 1). In the F plasmid origin, there are
two sites bound by DnaA and four 19-bp direct repeats
recognized by the monomers of RepE (Murotsu et al.,
1981; Matsunaga et al., 1995; Zzaman et al., 2004). Fewer
sites like this, located at two ends of the origin, were
identified in the origin of the pSC101 plasmid, which
contains just one site recognized by DnaA and three 21bp-long iterons bound by RepA (Churchward et al.,
1983), followed by two additional indirect repeats also
bound by initiator protein (Manen et al., 1992; Sugiura
et al., 1993; Ohkubo & Yamaguchi, 1995). The single
DnaA-box upstream of an AT-rich region was also identified in oric of plasmid R6K (MacAllister et al., 1991; Wu
et al., 1992). This E. coli plasmid contains, apart from
FEMS Microbiol Rev 36 (2012) 408–434
AT-rich region and the repeated sequences in DNA replication
oric, two other origins a and b, but both of them require
the presence of oric in cis for functioning (Crosa et al.,
1978; Kolter & Helinski, 1978, 1982; Shon et al., 1982;
Stalker et al., 1982; Mukherjee et al., 1988). The oric
required binding of the DnaA-box by DnaA and the
seven 22-bp iterons by the plasmid initiator protein p for
proper activity (Kelley & Bastia, 1992; Kelley et al., 1992;
Lu et al., 1998; Kruger & Filutowicz, 2000). It was shown
that binding of minimum five of seven iterons is required
for the origin activity (Kolter & Helinski, 1982) and that
the iterons’ binding occurs in a cooperative manner
(Bowers et al., 2007). Nucleotide changes in the iteron
sequences prevent binding of the p protein in vitro and
the plasmid replication in vivo (McEachern et al., 1985).
Moreover, mutants were isolated, containing changes
in the iteron sequences that reduced binding of p monomers or both monomers and dimers of the protein
(Kunnimalaiyaan et al., 2004). The sequences recognized
by the p protein are located downstream of the AT-rich
repeats (Fig. 1). Between the iterons and the site where
the helix melting occurs (Kruger & Filutowicz, 2003),
there is an additional site bound by the bacterial
IHF protein (Filutowicz & Appelt, 1988; Dellis et al.,
1996).
An unusual situation when compared with other analyzed plasmid origins could be observed in pPS10
and RK2 plasmids, where the sites recognized by chromosomal and plasmid initiators are located on the same side
of the AT-rich region. In the pPS10 plasmid, four 22-bp
iterons and one DnaA-box follow the AT region (Nieto
et al., 1992) and in plasmid RK2 the AT-rich region is
followed by five 17-bp iterons and four DnaA-boxes
(Stalker et al., 1981; Fuller et al., 1984; Konieczny et al.,
1997). Mutations within the pPS10 DnaA-box hampered
the replication in vivo, indicating the requirement for
DnaA binding within the origin (Nieto et al., 1992). The
single motif bound by the DnaA protein is located just
5 bp from the nearest iteron and its sequence differs in
the first nucleotide from the E. coli consensus (Giraldo &
Fernandez-Tresguerres, 2004). The RK2 DnaA-boxes also
contain one or two mismatches when compared with the
consensus and their binding by DnaA is necessary for
proper origin functioning. However, the requirement
for various DnaA-boxes present in oriV is host specific.
For example, the RK2 DnaA-box 3 and 4 should be
present in the origin during replication in E. coli and
P. putida but can be missed in P. aeruginosa (Doran et al.,
1999b). The sequence, number, orientation, and spacing
of the binding sites for the plasmid initiator protein TrfA
are also crucial for RK2 plasmid replication (Perri & Helinski, 1993). It was shown that the binding of TrfA to
just one iteron is unstable and probably a cooperative
binding occurs between the protein particles at the iterons
FEMS Microbiol Rev 36 (2012) 408–434
413
(Perri & Helinski, 1993). Similarly as it was observed for
E. coli oriC (Hsu et al., 1994 and see above), the spacing
between the particular regions of plasmid replication origin, and thus the proper helical phasing, is crucial for
effective replication. The insertion of 6 bp (approximately
half of one helical turn) between the AT-rich region and
iterons completely inactivated the origin, whereas the
11-bp insertion in the same region restored 55–78% of
replication activity compared with the activity obtained
for the wild-type (Doran et al., 1998). Typical iterons are
not found in R1 plasmid, which represents dissimilar
organization of origin when compared with the plasmid
described above. In the R1 replication origin, one inessential DnaA-box (Ortega-Jimenez et al., 1992) and two partially palindromic 10-bp sequences recognized by the
plasmid’s RepA protein are followed by three AT-rich
9-mers (Fig. 1; del Solar et al., 1998). The sites bound by
RepA are contained within the 100-bp region and it was
proposed that the binding of RepA causes a 100-bp DNA
loop formation, which is then filled with more RepA molecules and results in the opening of the AT-rich region
(Giraldo & Diaz, 1992; del Solar et al., 1998; Messer,
2002). Although the DnaA is not required for the replication initiation, the efficiency of R1 replication is increased
in the presence of the DnaA protein (Bernander et al.,
1991; Bernander et al., 1992). What’s interesting, in vivo
and in vitro experiments demonstrated that mutational
disruption of the DnaA-box sequence within the oriR
allowed decreased replication of the R1 plasmid from the
origin, whereas immuno-inactivation of the DnaA protein
totally abolished the process in vitro in the absence of the
DnaA-box sequence (Ortega-Jimenez et al., 1992). This
indicates that even though the utilization of DnaA is
favored for the R1 replication, the DnaA-box present in
the oriR seems to be redundant and in its absence the
DnaA recruitment to the origin may take place due to
DnaA–RepA interactions (Ortega-Jimenez et al., 1992).
In vivo experiments showed that deletion of the DnaAbinding site found downstream of the replication origin
of pBR322 plasmid does not have a significant effect on
the replication of the plasmid (Chiang et al., 1991).
It could be summarized that the AT-rich regions of
plasmid origins are usually followed by one or two
DnaA-boxes and precede the binding sites for plasmid
initiator protein. As in case of the chromosomal origins,
proper organization of the motifs within the origin is
important for the efficient functioning of replication proteins. The binding sites for initiator proteins should not
only be present in this region, but also be located in correct orientation in respect to each other, as very often the
changes in number, orientation, and even spacing
between the DnaA-boxes and iterons disturb replication
initiation.
ª 2011 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
414
Structure of the AT-rich region
Size and the internal stability of the AT-rich
region
The most distinguishable feature of an AT-rich region is
its high content of adenine and thymine residues when
compared with the overall average of an entire replication
origin. This depends on the base composition of a particular replicon and varies among chromosomal and plasmid origins. The region can be identified using in sillico
methods (see earlier paragraphs) based on sequence analysis and/or the internal thermodynamic stability of DNA.
However, defining the AT-rich region is sometimes rather
complicated since, as it was observed for example in
Streptomyces sp. (Jakimowicz et al., 1998), the region may
be dispersed and short AT stretches can be scattered
among other motifs. Nevertheless, typically there is no
bacterial origin and no DNA unwinding without an ATrich region.
In E. coli, the origin is composed in nearly 60% of
adenine and thymine residues, whereas its AT-rich
region consists of almost 77% of this type of bases.
However, the content may differ as much as in C. crescentus, where the general adenine and thymine content
in the origin is only about 34% and in the 40-bp-long
AT-rich region, it reaches 85% (Marczynski & Shapiro,
1992). Generally, the adenine–thymine richness of this
particular region varies from around 60% for P1 plasmid
origin, 71% in phage k (Schnos et al., 1988), almost
83% in R6K or 84% in pSC101 plasmids or R. meliloti’s
pSym-b megaplasmid origins, up to nearly 90% in case
of oriS of F plasmid. The length of an AT-rich region
within a replication origin can also be determined via
in sillico methods. It encloses a region of 40 bp, as for
example, in C. crescentus (Marczynski & Shapiro, 1992),
50–60 bp, as in E. coli, RK2 plasmid or Pseudomonas
origins (Bramhill & Kornberg, 1988b; Yee & Smith,
1990; Konieczny et al., 1997), to over 100 bp, as in the
case of R6K plasmid (Kruger & Filutowicz, 2003), within
the sequence of a replication origin. The pSC101 plasmid’s origin contains an asymmetric stretch of 80 bp,
rich in adenine and thymine residues (Churchward et al.,
1983; Manen & Caro, 1991). The high AT-content
results in the low thermodynamic stability of the region
which accounts for its role in the process of replication
initiation. At the AT-rich regions, the initial DNA helix
destabilization (opening) is induced by binding an initiator protein to its respective recognition sequences situated nearby. For chromosomal origins, these are DnaA
protein and DnaA-boxes and in case of most plasmids –
initiator proteins and iteron sequences. It is highly interesting that a protein interaction with specific DNA
ª 2011 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
M. Rajewska et al.
sequences opens the DNA helix at a site distant from
the protein’s recognition and binding sites. The extent of
the initial opening of an AT-rich region can only be
determined through biochemical analysis involving
experiments utilizing chemical modifications of thymine
residues by KMnO4 or P1 or S1 nucleases cleavage of a
ssDNA during replication initiation. The experiments
analyzing the exact DNA strand opening are scarce and
clear data can be found only for few chromosomal origins and some plasmids. However, when determined, the
extent of the opening does not differ too much in the
analyzed replicons and usually oscillates around 50 bp in
length. In the best analyzed replicon, E. coli, the DNA
fragment melting during initiation replication was determined to be c. 45 bp of the oriC sequence (Bramhill &
Kornberg, 1988a), although by the KMnO4 modifications
the initial melting was shown to encompass 26 bp (Gille
& Messer, 1991). Experiments concerning the opening of
the R6K’s oric in the presence of the p initiator, DnaA
and IHF proteins showed KMnO4 modifications within a
c. 55-bp fragment (Lu et al., 1998; Kruger et al., 2001)
of the 112-bp AT-rich region (Kruger & Filutowicz,
2003). Similar KMnO4 modification footprinting carried
out with the addition of TrfA initiator protein showed
the opening of the RK2 plasmid oriV origin within the
range of 46 bp on the top strand of DNA and 13 bp on
the bottom, which includes almost the whole AT-rich
region of 51 bp identified for the RK2 origin (Konieczny
et al., 1997). This is also the case of P1 plasmid, where
the opening was shown to presumably take place at
almost the whole extent of over 70-bp-long AT-rich
region (Park et al., 1998); however, here the adjacent
DnaA-box sequences were also included in the AT-rich
sequence. KMnO4 modifications occurred at the left end
of the region near the adjoining DnaA-boxes when the
bottom strand of the P1 plasmid DNA was analyzed.
When the analysis concerned the top strand, the modifications were detected in the middle of the AT-rich
region (Park et al., 1998). This might have been
observed due to the different base composition of the
particular strands. The analysis of S1 nuclease sensitivity
of the k phage DNA upon binding the k O protein to
iterons within the k origin demonstrated the presence of
an unwound ssDNA region of at least 23 bp and at most
49 bp of an AT-rich region adjacent to the iteron
sequences (Schnos et al., 1988). From these data, it can
be concluded that the initial opening of DUE is rather
no shorter than c. 20 bp and rarely longer than c. 50 bp,
which seems to be sufficient for the assembly of the prereplication complexes at the unwound site.
The pattern of thermodynamic stability is different for
various origins (see Fig. 2), nevertheless the AT-rich
region in most of the cases can be distinguished as the
FEMS Microbiol Rev 36 (2012) 408–434
AT-rich region and the repeated sequences in DNA replication
region of much lower free energy (DG) when compared
with the overall profile of the sequences surrounding it.
The regions contain some guanine and cytosine residues
and it is only natural that free energy will be accordingly
higher for those parts of the AT-rich sequences. In the
origins of E. coli (Fig. 2a), V. cholerae (Fig. 2d), C. crescentus (Fig. 2e) or F plasmid (Fig. 2j), the AT-rich
stretches form clear segments possessing noticeably lower
free energy when compared with the adjoining regions. In
other cases, as for example P1 or R6K plasmids (Fig. 2h
and k, respectively) the AT-rich region could not be
clearly distinguished among the origin sequences when
analyzing only the internal stability pattern. The fluctua-
415
tions and differences among the minima of free energy,
which can be observed when analyzing the thermodynamic stability patterns of particular origins, exist due to
the specific sequences of the regions and the existence of
direct repeats within the sequence. Their sequences determine the characteristic ‘intervals’ between points of the
local minima and maxima of free energy within some of
the repeats, as it can be observed in E. coli or V. cholerae
origins (Fig. 2a and d) or RK2 and pSC101 plasmids
(Fig. 2g and i). The internal stability pattern might also
be related to the helical periodicity of some of the repeats
within the AT-rich regions. This is the case in RK2 where
the local minima and maxima of the free energy could be
Fig. 2. Internal thermodynamic stability within selected replication origins’ fragments containing AT-rich regions. The stabilities were calculated
using WEBTHERMODYN software (http://gsa.buffalo.edu/dna/dk/WEBTHERMODYN/). The DNA sequence fragments taken for analysis were 200 bp
long. The red shading encloses the AT-rich regions with adjacent sequences. Black arrows depict repeats identified within the AT-rich regions of
the given origins. The sequence of the AT-rich regions and the repeats present within determine the characteristic ‘intervals’ between the points
of the local minima and maxima of the free energy of some of the replicons. Panels a-l present internal stabilities of selected chromosomal and
plasmid origins of replication.
FEMS Microbiol Rev 36 (2012) 408–434
ª 2011 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
416
ascribed to the distance of one helical turn. Most likely
the majority of the AT-rich regions of the prokaryotic
origins could contain repeats of various lengths. This
phenomenon is of great importance for the origin functioning and the formation of protein complexes during
replication initiation events (described in more detail in
the next paragraphs).
13-mers and other repeated sequences
Within the AT-rich regions of many prokaryotic origins
short repeated sequences have been identified; however,
their exact role was not explained up to date. Most of the
motifs in the AT-rich region are direct repeats, although
there are exceptions like RK2 plasmid’s oriV origin or
T. maritima’s where one of the motifs is inverted in relation to the other ones (Konieczny et al., 1997; Ozaki
et al., 2006) (Fig. 1). In E. coli, the AT-rich region is
present in the left part of the oriC origin and was shown
to contain three characteristic tandemly repeated
sequences, each 13-nucleotides long (Bramhill & Kornberg, 1988a). This length and the number of repeats seem
to be conserved among the chromosomal origins of replication, where the repeats were actually defined, as for
example in P. aeruginosa and P. putida (Yee & Smith,
1990), V. cholerae, Vibrio parahemolyticus and Vibrio vulnificus (Saha et al., 2004) (see Table 1). The E. coli
motifs, marked as L, M, and R (Bramhill & Kornberg,
1988a, b; Hwang & Kornberg, 1992a) are spaced with two
and three nucleotides, respectively. Upon the DnaA protein binding to the adjacent DnaA-box sequences the
region of the 13-mers unwinds, starting with the R motif
and successively proceeding toward the M and L ones
(Bramhill & Kornberg, 1988b). Initially it was assumed
that it is only the single-stranded structure and the helical
instability of the 13-mers that are important for the formation of the DUE and the origin activity. Escherichia coli
oriC deletion mutants lacking all three 13-mers were
unable to form an open complex and therefore were inactive (Bramhill & Kornberg, 1988b). Deleting the L 13-mer
from the oriC sequence led to an inhibition of origin
unwinding in the remaining two motifs, where no mung
bean nuclease hypersensitivity was detected in vitro and a
loss of the origin function in vivo was observed (Kowalski
& Eddy, 1989). However, the origin activity could be
restored by the insertion of a dissimilar DNA sequence in
the place of the deleted motif, suggesting that the L 13mer is not essential for oriC activity in vivo (Kowalski &
Eddy, 1989). Replacing this motif with a sequence containing higher GC content completely destroyed the origin activity (Asai et al., 1990). Recent studies
demonstrated that the deletion of the L 13-mer inactivates the heat-induced replication (HIR) and makes oriC
ª 2011 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
M. Rajewska et al.
inactive for cyclic replication in conditions of increased
growth temperature (Gonzalez-Soltero et al., 2006). It
was suggested that the strand opening for HIR initiation
occurs due to heat-induced destabilization of the L 13mer (Gonzalez-Soltero et al., 2006). The 13-mer motifs
possess a consensus sequence 5′-GATCTnTTnnTTT-3′
(Bramhill & Kornberg, 1988a). Experiments utilizing
sequence mutants showed that beside the low internal stability of the AT-rich region, also this specific sequence of
the motifs is important for the functionality of the origin
both in vivo and in vitro (Hwang & Kornberg, 1992a). Of
the three 13-mer repeats, the sequence of the R one seems
to be the most essential for origin function, as even single
mutations or one base insertions are sufficient to disable
the origin activity (Bramhill & Kornberg, 1988b; Asai
et al., 1990; Hwang & Kornberg, 1992a, b). The E. coli
oriC AT-rich region also contains, apart from the
13-nucleotide motifs, an AT cluster situated at the leftmost part of the origin adjacent to the L 13-mer, that
determines this border of origin (Asai et al., 1990).
The 13-mers found in Pseudomonas origins are packed
less tightly than those of E. coli oriC and spaced with
sequences of 11 and 12 bp in length (Yee & Smith, 1990).
The sequence composition of the P. putida repeats
accounts for the formation of local minima of thermodynamic instability (Fig. 2c), where the lowest energy points
are at the terminal nucleotides of the M and R repeats
that contain more AT residues than the beginning of the
sequences (see Table 1). The sequence of the Pseudomonas repeats differs from that in E. coli consensus in
respect to the GATC motif found within all 13-mers in
oriC. Only the P. putida L 13-mer contains that motif at
the beginning of its sequence. When the whole sequence
is compared, the repeats of pseudomonads differ from
the E. coli consensus in as much as four to even seven
nucleotide positions. The AT-rich regions in V. cholerae,
V. parahemolyticus and V. vulnificus contain sets of three
13-mer repeats L, M, and R, bearing close sequence consensus with the respective motifs of E. coli oriC; however,
only the R repeats show the highest accordance with the
oriC 13-mer consensus as well as with its length (Saha
et al., 2004). This could be expected as earlier studies
concerning replication in E. coli showed that the R 13mer’s sequence and length are essential for the proper initiation of replication (see earlier paragraph). Besides the
13-mer motifs, the AT-rich region of Vibrio origins also
contains a short tract, AT cluster, preceding them and
composed of adenine and thymine residues only (Saha
et al., 2004), as it is observed in E. coli oriC. The 40-bplong AT-rich region of C. crescentus contains a sequence
that is very similar to the 13-mer motifs of E. coli (Marczynski & Shapiro, 1992). When the sequences spacing
the E. coli 13-mers are taken into account, the C. crescentus
FEMS Microbiol Rev 36 (2012) 408–434
AT-rich region and the repeated sequences in DNA replication
417
Table 1. Repeated sequences within the AT-rich regions of selected replication origins
The table lists sequences of the repeated sequences present within AT-rich regions of selected chromosomal and plasmid origins of replication.
The distances between respective repeats are given in number of bp. The potential SeqA binding and methylation sites possessing GATC
sequence are enclosed in green boxes. In the bacterial origins there are typically up to three repeats within the AT-rich regions, usually of 13 nucleotides in length and a sequence similar to the consensus determined for Escherichia coli oriC 13-mers. In the chromosomal repeats, distinctive
core within the sequence (boxed in red), containing the highest content of adenine and thymine residues can be distinguished. For the plasmid
repeats, their length varies more and typically their entire sequence consists mostly of adenines and thymines.
FEMS Microbiol Rev 36 (2012) 408–434
ª 2011 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
418
13-mer matches 13 of 15 bp in the E. coli L, M, and R
13-mers (Marczynski & Shapiro, 1992). Therefore, the
motif may have similar functional significance as its
equivalents in E. coli. Concluding from the internal stability pattern of Cori fragment, containing the sequence of
the AT-rich region where the 13-mer is found in the
region of the lowest thermodynamic stability (Fig. 2e), it
can be assumed that it is the place where DNA helix
unwinding is initiated during replication initiation. At the
replication origin of B. subtilis, two AT-rich regions were
defined, one containing three repeats of 16 nucleotides in
length and the other consisting of a 27-bp cluster (Moriya
et al., 1985; Yoshikawa & Wake, 1993). The importance
of the 16-mers was not determined, as KMnO4 modifications upon binding of the DnaA protein to the origin
demonstrated, that it is the 27-mer that serves as the
DUE for the Bacillus origin (Moriya et al., 1994; Krause
et al., 1997). In T. maritima, three 9-nucleotide-long
sequences were found within one of its origin’s AT-rich
regions, marked AT-1, AT-2, and AT-3, sharing a consensus of 5′-TATTATTnA-3′ (Ozaki et al., 2006) and consisting almost in 93% of only adenine and thymine residues.
The comparative analysis of T. maritima AT sites pointed
out some sequence homology with the E. coli 13-mers
and the corresponding archaeal and eukaryotic sequences
(Ozaki et al., 2006). Only two of those repeats, AT-2 and
AT-3, are sufficient for the functionality of the T. maritima minimal origin. Binding an ATP-tmaDnaA (T. maritima homologue of the DnaA protein) at the tma-oriC
leads to duplex unwinding and the formation of an open
complex at the 9-mer region, depending on the temperature and superhelical tension of a plasmid containing the
tma-oriC. No 13-mers similar to those of E. coli were
found in oriC of Streptomyces; however, five short AT-rich
sequences are distributed among the sequences of DnaAboxes at the origins of those bacteria (Jakimowicz et al.,
1998). Based on the above finding, it can be observed that
in the bacterial origins the repeats within the AT-rich
regions are usually 13 nucleotides long and possess rather
high sequence similarity with consensus similar to that
determined for E. coli oriC 13-mers (see Table 1). A distinctive core consisting mostly of adenines and thymines
can be distinguished in the sequence of the chromosomal
repeats (see Table 1, boxed in red). Most often there are
three direct repeats in the region that play a direct role in
the open complex formation at the origin during the replication initiation.
Repeated sequences were also identified in the AT-rich
regions of plasmid origins and phage k, in up to five copies per origin. Those repeats seem to contain mostly adenine and thymine residues, which makes them c. 80 to
even 100% AT-rich. It is rather difficult to determine a
consensus sequence for the repeats found among different
ª 2011 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
M. Rajewska et al.
plasmids as they are of various lengths but a consensus
can be established for the repeats within a particular origin. Some of the repeats are shorter than the 13-mer
motifs of oriC, just like the 7-mers of P1 plasmid (Chattoraj et al., 1985; Abeles et al., 1990; Brendler et al.,
1991a, b; Park et al., 1998), 8-mers at oriS of F plasmid
(Wei & Bernander, 1996), 9-mers in R1 (Wei & Bernander, 1996) and 10-mers in R6K plasmids (Stalker et al.,
1979; Bramhill & Kornberg, 1988a) or 11-mers in orik
(Schnos et al., 1988), others preserve the 13-nucleotide
length as in the case of pSC101 plasmid (Wei & Bernander, 1996; Miller et al., 2003) or oriV origin of RK2
(Stalker et al., 1981; Konieczny et al., 1997) (see Table 1).
In the latter, four 13-nucleotide repeats can be distinguished within the AT-rich region: L, M1, M2, and R,
bearing a consensus sequence 5′-TAAACnTTnTTTT-3′
(Konieczny et al., 1997) similar to that defined for oriC
(Bramhill & Kornberg, 1988a). In those repeats, as in E.
coli, the most stringently conserved are the nucleotides at
the beginning and the end of each motif: TAA and TTTT
(see Table 1). Those initial and terminal nucleotides
account for the segments of the lowest internal stability
within the AT-rich region and as the middle part of each
13-mer shows higher G/C content, characteristic fluctuations of the thermodynamic stability can be observed for
this part of the region (see Fig. 2g). The AT content in
the RK2 13-mers reaches almost 80%. They are tightly
packed and their sequences partially overlap; three of
them, L, M1, and R, are direct and situated on the top
strand of DNA, the fourth, M2, is reversed and placed
on the bottom strand (Konieczny et al., 1997). This
arrangement of the repeats is crucial for origin functioning as introducing insertions between them disabled
origin’s activity in vivo (Kowalczyk et al., 2005). The
in vivo and in vitro experiments utilizing a group of
mutants of the RK2 oriV AT-rich region also demonstrated that its role in replication initiation does not
ensue only from its low inner thermodynamic stability.
It was shown that the position of each 13-mer within
the AT-rich region is critical for the activity of the
plasmid’s origin, as swapping or reversing the motifs had
a negative effect on origin function (Kowalczyk et al.,
2005).
As it was observed for 13-mers in the chromosomal
origins, the number and length of the repeats within the
AT-rich region of an individual plasmid origin possibly
also plays an important role in the origin’s activity. For
the RK2 plasmid, it was demonstrated that the deletion
of one of the 13-mer repeats present in the AT-rich
region disabled oriV activity in vivo (Kowalczyk et al.,
2005). In R. meliloti pSym-b megaplasmid, only one copy
of 13-mer was defined, varying from the E. coli consensus
sequence in three positions (Margolin & Long, 1993).
FEMS Microbiol Rev 36 (2012) 408–434
AT-rich region and the repeated sequences in DNA replication
The AT-rich region of the origin of pSC101 contains two
13-mer repeats (Manen & Caro, 1991) that are homologous to those found in oriC of E. coli. The c origin of
R6K plasmid contains three repeats of 10-nucleotide-long
sequences (Stalker et al., 1979; Bramhill & Kornberg,
1988a), nearly 100% AT-rich, whose content is higher
than that of the entire AT-rich region defined at the origin. The repeats, L and M 10-mers, are spaced with relatively long sequence of 29 bp and the distance between M
and R is 9 bp long. The lengths of the repeats and the
spacers in between them approximately multiply the
length of one helix turn (c. 11 bp) which might be significant for the DNA destabilization during replication initiation from the c origin or for nucleoprotein interactions
within the AT-rich region. It was demonstrated that the
R6K plasmid’s initiator protein p binds as a dimer to the
sequences within the AT-rich region at higher concentrations and possibly serves this way as a negative regulator
of primer formation at the site (Kruger & Filutowicz,
2000). In some cases, determining the repeats is more
complicated and different publications give different data
concerning the same replicon. For example, the work of
Wei and Bernander (Wei & Bernander, 1996) identifies
four highly AT-rich repeats of 8 bp in length (see
Table 1) within the origin of plasmid F. They account for
the region of the lowest thermodynamic stability when
compared with the surrounding sequences (see Fig. 2j).
However, Kawasaki et al. (1996) indicate a 13-nucleotidelong sequence 5′-GATCTTCTTTTTT-3′, overlapping two
of the four 8-mer motifs (M2 and R), and matching the
oriC 13-mer consensus at 11 positions, as the site of
localized DNA melting upon binding of the RepE, HU
and DnaA proteins to the origin, as it was shown by
experiments utilizing P1 nuclease cleavage of ssDNA. Further DNA unwinding at the F plasmid origin proceeds
toward the left side of the AT-rich region, where the
putative L and M1 motifs were suggested by the other
group. Differences also concern the origin of P1 plasmid,
where various works identify five tandem 7-nucleotide
repeats (Chattoraj et al., 1985; Abeles et al., 1990; Brendler et al., 1991a, b) within the central part of the origin,
others (Park et al., 1998) extend the repeats’ sequences to
10 bp. Although they are not designated as AT-rich (the
adenine and thymine content of each individual repeat
oscillates between 45% and 55%), the DNA strand melting takes place within the repeats containing region (Park
et al., 1998). Experiments utilizing mutants of the RK2
oriV AT-rich region demonstrated that the repeats are
also origin specific. Those mutants where the sequence of
a particular 13-mer was substituted with a respective
sequence of E. coli oriC showed a total lack of activity
in vivo and a significantly reduced replication activity
in vitro (Kowalczyk et al., 2005).
FEMS Microbiol Rev 36 (2012) 408–434
419
Mutagenesis at the AT-rich repeats was conducted
within different plasmid replicons. Single-base substitutions in the P1 plasmid’s AT-rich repeats demonstrated
that the sequence of the 7-mers is essential for the functionality of the P1 origin (Brendler et al., 1991a, b). Those
repeats contain GATC motifs (see description in the paragraph below) within their sequence. The negative effect of
mutations in other positions was also observed when
GATC motifs were left intact. For each of the repeats, at
least one single-base change that had a deleterious effect
on replication was found. The phenotype effects of mutations in the first 7-bp repeat (5′-AGATCCA-3′) showed
that 6 of 7 bp are important for origin function, whereas
changes in the last position of the motif had little or no
effect. The integrity of the sequence of the 7-nucleotide
motifs seems to be crucial for the origin activity but neither the sequence nor the length of the 3- to 6-bp spacing
between the motifs is very important, although the
requirement of minimal spacer length was suggested
(Brendler et al., 1991a, b). In the case of k origin’s ATrich region studies concerning the prepriming step of k
DNA replication initiation and the origin opening demonstrated that a point mutation in one of the 11-mers,
adjacent to the first iteron sequence of the origin, where
a G/C was substituted with a T/A pair, had an effect on
AT-rich region sensitivity to S1 cleavage which was found
remarkable as the mutation increases the A/T content of
the region (Schnos et al., 1988). Point mutations within
the AT-rich 13-mers of the RK2 plasmid also negatively
influenced the origin activity, decreasing it to 75 or even
to 0.01% when compared with the activity of the wildtype origin (Kowalczyk et al., 2005). What was also extremely interesting here, even the A/T or T/A substitutions
in the conserved initial or terminal sequences of each
13-mer, not affecting the pattern of the thermodynamic
stability of the region, caused a complete loss of activity
of the origin (Kowalczyk et al., 2005). This suggests that
the position of the adenine and thymine residues on each
DNA strand may be critical for the interactions of proteins involved in the plasmid’s replication initiation
within this region. Changes simultaneously affecting the
sequences of putative DnaA-box motifs within this region
showed that their sequence is also important to the origin’s activity (Kowalczyk et al., 2005); however, the
DnaA–DNA interaction within this region still remains
elusive. The activity of the oriV AT-rich region’s mutants
was similar in the in vivo experiments in E. coli and
P. aeruginosa, except for two of the mutants having
changes within the M1 13-mer, which being inactive in
E. coli could replicate in P. aeruginosa (Kowalczyk et al.,
2005). It is possible that this particular motif is important
for the plasmid’s replication in E. coli but not in
P. aeruginosa. This raises the possibility that host factors
ª 2011 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
M. Rajewska et al.
420
can specifically interact within the AT-rich region of the
plasmid origin. Moreover, it could be that the subtle
changes of the internal stability resulting from the
sequence change could affect replication activity in one
host but not the other. All in all, the above data demonstrates that the repeats found in the plasmids’ AT-rich
regions play an essential role in the DNA replication initiation. Their length, number, and spacing are individual
for each origin and the sequence of each is substantial for
origin functioning.
Motifs overlapping the AT-rich region’s
repeated sequences
Apart from the direct repeated sequences, there are other
motifs that can be identified within the AT-rich regions
of prokaryotic replication origins that play a crucial role
in the process of replication initiation or are involved in
its control (see Table 2). In the AT-rich region of E. coli
oriC, 6-nucleotide-long sequences of ATP-DnaA-boxes
were identified (Speck & Messer, 2001), possessing a consensus sequence 5′-AGATCT-3′. Those motifs overlap the
13-mers L and M found in oriC and are the recognition
sites for the ATP form of the DnaA initiator protein
(Speck et al., 1999). The interaction of the ATP-DnaA
with these sequences after the formation of a singlestranded structure was postulated to be important during
the process of open complex formation within the oriC
(Speck & Messer, 2001). Possible ATP-DnaA-box
sequences can be found in the AT-rich regions of Vibrio
species origins which are similar in structure to those of
E. coli oriC (Saha et al., 2004). Moreover, a single AGAT
CT sequence was found in the origins of C. crescentus
and the F plasmid; however, no data determine the function of those sequences in DNA replication of those replicons. Other specific motifs, TTGT and TTATT, within
the E. coli ssDNA of M and R 13-mers were identified to
interact with the ATP-DnaA protein (Ozaki et al., 2008).
No ATP-DnaA-boxes that would be similar to those in
oriC are present within the RK2 13-mers (Speck &
Messer, 2001; Kowalczyk et al., 2005). However, on the
borders of the 13-mers M1 with M2 and M2 with R,
sequences of putative DnaA-boxes were identified
(Kowalczyk et al., 2005), where the DnaA protein may
Table 2. Proteins interacting or suspected to interact within the AT-rich regions
Protein
Replicon
DnaA
DnaA
DnaA
DnaB
E. coli
E. coli
V. harveyi
E. coli
DnaC
DnaG primase
Pol III holoenzyme
core
b-ring
SSB
P
SeqA
IciA
E. coli
E. coli
E. coli
Π
Fis
ArcA
IHF
HobH
DpiA
CspA; CspE
CspB; CspC; CspD
E. coli
Phage k
E. coli
E. coli; F;
R1
R6K
R6K
E. coli
R6K; pSC101
E.
E.
E.
E.
B.
coli
coli; pSC101
coli
coli;
subtilis
Recognized sequence/
binding specificity
References
AGATCT
TTGT; TTATT
AGATCG
ssDNA; M/R 13-mers;
bottom strand
Cryptic ssDNA binding
ssDNA
ssDNA
Speck & Messer (2001)
Ozaki et al. (2008)
Messer (2002)
LeBowitz & McMacken (1986); Jezewska et al. (1996); Weigel &Seitz (2002)
Learn et al. (1997)
Wickner (1977); Rowen & Kornberg (1978)
Kelman & O’Donnell (1995)
ssDNA
Cryptic ssDNA binding
GATC
dsDNA; 13-mers
Meyer & Laine (1990)
Learn et al. (1997)
Brendler et al. (1995); Slater et al. (1995)
Thony et al. (1991); Hwang & Kornberg (1992a, b); Wei & Bernander (1996)
dsDNA
dsDNA
dsDNA
TTTAAGTTGCTGATT;
CCACAACTCAAA
dsDNA
dsDNA; 13-mers
ssDNA
ssDNA
Kruger & Filutowicz (2000)
Wu et al. (1996)
Lee et al. (2001b)
Filutowicz & Appelt (1988); Levchenko & Filutowicz (1996); Datta et al.
(1999)
Herrick et al. (1994)
Miller et al. (2003)
Jiang et al. (1997); Phadtare & Inouye (1999); Yamanaka et al. (2001)
Graumann & Marahiel (1994); Graumann et al. (1997); Jiang et al. (1997);
Phadtare & Inouye (1999); Yamanaka et al. (2001)
In the table, proteins interacting or suspected to interact with the sequences of the AT-rich regions of replication origins are listed. If a specific
sequence was identified to be bound by a protein, it is given in the table (see details in the text). Otherwise only ssDNA or dsDNA interaction is
implied.
ª 2011 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
FEMS Microbiol Rev 36 (2012) 408–434
AT-rich region and the repeated sequences in DNA replication
interact with DNA, as it was suggested previously (Gaylo
et al., 1987; Doran et al., 1999a, b). The exact role of the
DnaA-box sequences located within DUE is being investigated. It is difficult to comprehend if the ATP-DnaAboxes are universal motifs that could be found in more
origins or if they are unique only for some. However,
most likely the interaction of replication initiators with
specific motifs within an AT-rich region of an origin
could be widespread.
The sites recognized by Dam methyltransferase (Geier
& Modrich, 1979; Slater et al., 1995) were found overlapping the repeated sequences but also present in the adjacent sequences of the AT-rich regions. Those sites are the
short GATC motifs, where methyl group is introduced at
the adenine residues (Geier & Modrich, 1979). The
methylated GATC sites become hemimethylated during
the DNA replication and as such are recognized by a
SeqA protein (Brendler et al., 1995; Slater et al., 1995)
that is involved in sequestration of the newly synthesized
DNA (Lu et al., 1994), preventing premature DNA rereplication in vivo. SeqA binds to an origin tandemly as a
dimer (Lee et al., 2001a; Guarne et al., 2002; Han et al.,
2003; Guarne et al., 2005; Kang et al., 2005) and was
found to negatively regulate DNA replication by blocking
the GATC sequences within the DnaA-boxes of E. coli
oriC and preventing DnaA binding to those sites (Taghbalout et al., 2000; Nievera et al., 2006). The GATC sites
that are found within the E. coli origin’s AT-rich region
at the beginning of each 13-nucleotide sequence are also
recognized by SeqA. The highest affinity of the SeqA protein was shown at the GATC motif located within the
oriC L 13-mer (Kang et al., 1999) but strong binding was
shown also for the GATC motifs within M and R 13mers. Here SeqA inhibits the formation of the open complex at the AT-rich region of the origin (Torheim &
Skarstad, 1999). The SeqA recognition sequences are
found in similar positions of the repeats identified within
AT-rich region of P. putida, V. cholerae, V. parahemolyticus, V. vulnificus, C. crescentus or R. meliloti, but also in
some plasmid AT-rich sequences as for example F,
pSC101 or P1 (see Table 1). In F plasmid, they overlap
the 7-nucleotide repeated motifs and both the GATC
sequence and its methylation are important for the functionality of the F origin in vivo (Brendler et al., 1991a, b,
1995; Abeles et al., 1993). No GATC sequences were
found within the AT-rich region of the RK2 oriV origin,
which excludes the 13-mers as sites for SeqA interaction.
As GATC motifs are noticeably more frequent in the ATrich regions of chromosomal origins of replication, their
role in the regulation of replication initiation and chromosome segregation is of great importance.
Specific interactions within the 13-mer sequences of
oriC were reported for the IciA protein, which is an
FEMS Microbiol Rev 36 (2012) 408–434
421
inhibitor of chromosome replication initiation (Hwang &
Kornberg, 1990, 1992b; Thony et al., 1991). Binding the
IciA dimer to DNA at the 13-mers causes an early
inhibition of the unwinding process at the AT-rich region
by DnaA and HU proteins, preventing the replication
initiation (Hwang & Kornberg, 1990, 1992a). The introduction of sequence changes within the L 13-mer may
lead to an increase or decrease of IciA affinity toward the
13-mers, therefore inhibiting the process at a different
level (Hwang & Kornberg, 1990). This also suggests that
there might exist specific sequences recognized by the
protein within the 13-mers. IciA does not inhibit the
binding of the DnaA and IHF proteins to their respective
sequences (Hwang & Kornberg, 1992a). However, once
the 13-mers are opened due to the DnaA protein activity,
IciA no longer affects the DNA replication from the oriC
origin (Hwang & Kornberg, 1990). EMSA and DNase I
footprint analyses showed that sites recognized by the
IciA protein are also found at the F and R1 plasmid origins in their repeat-containing AT-rich regions, as it was
observed for E. coli oriC (Wei & Bernander, 1996) and
possibly play a similar role in replication initiation. No
data were found for the presence of IciA-recognized
sequences within the AT-rich regions in other origins;
however, such possibility cannot be excluded without
proper biochemical analysis.
Sites recognized also by other proteins were shown to
exist within the oriC AT-rich region. For example, a site
for binding the ArcA protein that, among other
sequences, interacts with the 13-mers and blocks the
open complex formation and therefore the replication
initiation. The protein binding does not affect the process
once the ssDNA structure is formed (Lee et al., 2001b).
Sequences within the 13-mer region were also reported
to bind a putative E. coli membrane protein, HobH, that
seems to be involved in postreplication oriC binding to
the membrane at the DNA hemimethylated state, preventing re-initiation (Herrick et al., 1994). Binding the
13-mers at the AT-rich regions of the oriC and pSC101
plasmid origins by a DpiA protein results in perturbed
DNA replication and plasmid inheritance destabilization;
also, the DpiA overexpression and binding to oriC
induces bacterial SOS response (Miller et al., 2003). The
sequences at the unwound AT-rich region of oriC were
found to take part in the interaction with another factor –
the CspD protein, belonging to the cold shock proteins
family. Its binding to the ssDNA was shown to effectively
inhibit both the initiation and elongation stages of the
minichromosomal DNA replication in vitro (Yamanaka
et al., 2001); however, the binding occurred without
apparent sequence specificity. The feature of ssDNA
binding of CspD is shared by other Csp proteins in
E. coli: CspA, CspB, CspC, and CspE (Jiang et al., 1997;
ª 2011 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
422
Phadtare & Inouye, 1999) and B. subtilis: CspB, CspC,
and CspD (Graumann & Marahiel, 1994; Graumann
et al., 1997).
The binding sites for nucleoid-associated proteins
(NAPs) were also identified within AT-rich region of some
replication origins. For example, Fis (factor for inversion
stimulation) binding sites, which are present outside the
AT-rich region of E. coli oriC (Gille et al., 1991), were
found in the sequence of the AT-rich region of the R6K
plasmid. The plasmid replication depends on Fis when
copy-up variants of R6K p protein and the Penr marker
were simultaneously used (Wu et al., 1996). In case of two
plasmids, R6K and pSC101, sites for binding the hostencoded DNA bending protein IHF (integration host
factor) were localized within the AT-rich regions of their
respective origins (Filutowicz & Appelt, 1988; Levchenko
& Filutowicz, 1996; Datta et al., 1999). The IHF stimulates
the unwinding of plasmid origins and E. coli oriC
(Bramhill & Kornberg, 1988b; Hwang & Kornberg, 1992a;
Kruger et al., 2001). It has been demonstrated that Fis
protein suppresses IHF as well as DnaA interaction within
the E. coli oriC and negatively affects replication initiation
(Ryan et al., 2004). Another factor interacting with ssDNA
and functionally affecting its structure is the ssDNA-binding protein (SSB), which interacts with the open region of
an origin and is involved in replication complex assembly
(Meyer & Laine, 1990).
Apart from the proteins described above also other factors were identified to be crucial for the regulation of
replication activity of prokaryotic origins. Some of them
influence origin opening at the AT-rich region; however,
none of them was demonstrated to interact directly with
the motifs found in this region. The RIDA mechanism
(regulatory inactivation of DnaA) (Katayama et al.,
1998), stimulated by Hda protein and b-ring (Katayama
et al., 2010), prevents overinitiation of DNA replication
by converting the active ATP-DnaA form to its inactive
ADP-DnaA form (Camara et al., 2005). The DnaA inactivation occurs due to direct interaction of Hda with the
b-clamp through a specific motif at its N-terminus (Kurz
et al., 2004). This process, together with the dnaA gene
autoregulation, acts to ensure homeostatic maintenance
of the E. coli chromosome (Riber et al., 2006). Rob protein binds the right side of oriC, the R2, R3, and R4
DnaA-boxes, which may influence the formation of
nucleoprotein structure in this region (Skarstad et al.,
1993). Cnu (oriC-binding NAP) could possibly play a role
in proper activity of oriC (Kim et al., 2005). DiaA
(DnaA-binding protein) binds DnaA complexed with
DnaA-box and stimulates the formation of an open complex (Ishida et al., 2004) and directly the formation of
the ATP-DnaA multimers on oriC ensuring timely replication (Keyamura et al., 2009).
ª 2011 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
M. Rajewska et al.
Relevance of repeated sequences within
AT-rich region for DNA replication
initiation
AT-rich region repeated sequences and origin
opening
The high content of adenine and thymine residues and
consequent low internal stability of the AT-rich region
facilitates DNA helix destabilization. It was observed
in vitro that to some extent, the DUEs have a tendency
toward spontaneous opening (breathing) without replication initiation factors (Polaczek et al., 1998). The E. coli
oriC unwinding can occur in either the presence or the
absence of initiation proteins, as detected by a singlestrand specific nuclease, P1 (Polaczek et al., 1998). This
suggests that oriC unwinding could be a spontaneous
event determined solely by the DNA sequence containing
repeated motifs contributing to the specific internal stability pattern with periodical local minima of free energy
(see paragraphs above). Therefore the replication proteins
may function only to stabilize the ssDNA structure.
Experiments conducted on other systems revealed that the
DNA helix unwinding could be detected only in the presence of the replication initiation proteins (Schnos et al.,
1988; Mukhopadhyay et al., 1993; Kawasaki et al., 1996;
Konieczny et al., 1997; Kruger et al., 2001; Kruger & Filutowicz, 2003). For example, in vitro tests with KMnO4
clearly demonstrate that during the initiation of RK2 plasmid’s replication, the opening of the AT-rich region is
induced by the plasmid replication initiation protein TrfA
and is only enhanced or/and stabilized by host DnaA
(Konieczny et al., 1997). Moreover, a strict requirement
of histone-like proteins indicates that the appropriate superhelicity of DNA within DUE is critical for its opening.
The necessity for HU or IHF proteins was reported for
E. coli oriC (Kano et al., 1991; Hwang & Kornberg, 1992a;
Ryan et al., 2002), phage k (Mendelson et al., 1991), plasmids F (Ogura et al., 1990; Kawasaki et al., 1996; Zzaman
et al., 2004), P1 (Ogura et al., 1990; Mukhopadhyay et al.,
1993; Park et al., 1998; Fekete et al., 2006), R6K (Dellis &
Filutowicz, 1991; Dellis et al., 1996; Lu et al., 1998;
Kruger et al., 2001; Abhyankar et al., 2003) and RK2
(Konieczny et al., 1997). Although the spontaneous destabilization of the AT-rich part of the origin is possible, no
functional origin opening leading to the formation of replication complex and the initiation of DNA replication
was observed without the initiator proteins. Therefore the
analysis of the nucleoprotein complexes formed at the
replication origin is fundamental to our understanding of
the mechanism of the initial origin opening.
It is thought that the initial complex at E. coli oriC
includes 5–20 DnaA molecules and it forms a wrapped
FEMS Microbiol Rev 36 (2012) 408–434
AT-rich region and the repeated sequences in DNA replication
nucleoprotein structure (Kornberg & Baker, 1992; Carr &
Kaguni, 2001; Messer et al., 2001). A similar multimeric
form called O-some consisting of the kO protein and
iterons was identified during the bacteriophage lambda
DNA replication initiation (Dodson et al., 1989). The formation of the E. coli oriC initial complex requires sequential DnaA protein binding to the individual DnaA-box
sequences, located within the origin’s sequence (Margulies
& Kaguni, 1996; Weigel et al., 1997). While ATP-DnaA
and ADP-DnaA are both able to form nucleoprotein
complexes at oriC, the ATP-DnaA is required for the
DUE opening (Bramhill & Kornberg, 1988a, b; Sekimizu
et al., 1988; Ozaki et al., 2008). The DnaA-boxes of the
chromosomal replication origins, as well as the plasmids’
iterons, are usually located on one side of AT-rich
sequences (see paragraph above and Fig 1). This indicates
that the initial complex, consisting of Rep proteins and
the wrapped DNA, is formed in the proximity but outside of the DUE element. Experiments, demonstrating
that the distance and helical spacing between the Rep
binding sites and an AT-rich region is critical for origin
activity (see paragraphs above and Hsu et al., 1994;
Doran et al., 1998), raised the possibility that the DUE
can directly interact with a nucleoprotein structure
formed by Rep proteins.
The last 10 years brought new data on the mechanism
of the initial DNA unwinding and highlighted the contribution of the DNA sequence of the AT-rich region to the
open complex formation. Mutational analysis revealed
that the E. coli oriC activity requires DnaA interaction
with both strong affinity sites (R1, R2, and R4) and low
affinity sites (R3, R5, and I-sites) and also that both ATPDnaA and ADP-DnaA forms interact at those sites (Leonard & Grimwade, 2005; Grimwade et al., 2007). The discovery of the ATP-DnaA-boxes within the AT-rich region
of the oriC origin of E. coli (Speck & Messer, 2001)
pointed out that DnaA can interact within the specific
sequences that are present at the AT-rich region. SPR
analysis of the DnaA nucleoprotein complexes clearly
demonstrated that the ATP-DnaA binds ssDNA containing ATP-DnaA-box sequences (Speck & Messer, 2001;
Messer, 2002). It was proposed (Speck & Messer, 2001)
that initially DnaA protein binds with high affinity to
dsDNA oriC DnaA box R1 and that this complex serves as
an anchor for a cooperative binding of ATP-DnaA to the
ATP-DnaA-boxes. This would position the protein to the
region of unwinding. The ssDNA resulting from the DUE
unwinding would then be stabilized by cooperative binding of ATP-DnaA. Crystallographic data on DnaA proteins, isolated from Aquifex aeolicus and T. maritima, and
crystal structures of Rep proteins of plasmids F, pPS10,
P1, R6K as well as the structural data obtained via bioinformatics’ approaches (Sharma et al., 2004; Pierechod
FEMS Microbiol Rev 36 (2012) 408–434
423
et al., 2009) elucidated general mechanisms underlying the
DNA replication initiation in bacteria. It became clear that
both the chromosomal DNA replication initiators, DnaA
homologous proteins and the plasmid replication initiators, Rep proteins, form oligomers when interacting with
their binding sites localized at the replication origin.
Structural analysis of the F plasmid’s RepE (Komori et al.,
1999) and the A. aeolicus DnaA (Erzberger et al., 2002)
was consistent with previous observations showing that
the replication initiators induce DNA bending, when
interacting with a DNA particle (Mukherjee et al., 1985;
Stenzel et al., 1991; Murakami et al., 1997). The repeated
sequences, iterons, and DnaA-boxes contribute to that
specific nucleoprotein structure. It must be pointed out
that due to the structural variations among the DnaA
homologues and the Rep proteins, the nucleoprotein complexes they form with DNA differ. The structure analysis
of the A. aeolicus DnaA protein (Erzberger et al., 2002)
revealed that the ATP-binding domain, including two
Walker motifs for nucleotide binding and hydrolysis,
belongs to the AAA+ superfamily (Neuwald et al., 1999;
Maurizi & Li, 2001; Ogura & Wilkinson, 2001). The protein’s C-terminal domain consists of a classic helix-turnhelix motif and an extra loop that could be in contact
with DNA (Erzberger et al., 2002). Based on the obtained
structural data, it was proposed that the DnaA oligomer
could conceivably accommodate either a closed ring or a
helical filament arrangement of monomers. The DUE
opening may occur spontaneously through a local strain
induced by an assembly of the nucleoprotein complex in
the presence of ATP (Erzberger et al., 2002). In a recent
study, Berger’s group reported that DnaA utilizes at least
two different oligomeric conformations and that it plays
distinct roles in controlling the progression of the replication initiation (Duderstadt et al., 2010). The structural
basis for a link between the dsDNA binding, the ability to
form oligomers and the interaction with ssDNA in ATPdependent manner was established. Moreover, experiments on the T. maritima DnaA showed the multimer
ATP-tmaDnaA-dependent formation of an open complex
at the replication origin (Ozaki et al., 2006). ATP is not
required for the E. coli DnaA binding to the DnaA-boxes
and the formation of multimeric complex at the oriC;
however, it is essential for the protein interaction with the
ATP-DnaA-boxes localized within the AT-rich region. The
interaction of DnaA proteins with specific motifs located
within the ssDNA of the AT-rich region is suggested to be
a common mechanism for the origin opening of bacterial
chromosomes.
Understanding the open complex formation at plasmid
origins is more complex as two initiator proteins are
involved during plasmid replication initiation. In contrast
to the DnaA homologues, the Rep proteins do not contain
ª 2011 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
424
AAA+ domain and consist of two winged helix (WH)
domains, which are a large family of DBDs found also in
eukaryotic organisms (Giraldo, 2003). The WH domains
are composed of a bundle of three a-helices and an antiparallel b-sheet (Gajiwala & Burley, 2000) that facilitate
Rep binding to the direct repeated sequences at plasmid’s
replication origin. No ATP binding sites were identified in
the Rep structure and, in contrast to the chromosomal
DNA replication initiation, no strict ATP requirement was
observed for plasmids’ origin opening (Mukhopadhyay
et al., 1993; Kawasaki et al., 1996; Lu et al., 1998; Park
et al., 1998). Although the Rep DNA binding and the
DUE opening at plasmid origins are not ATP-dependent,
the specific binding of Rep to the iterons at plasmid origins establishes oligomeric nucleoprotein complex similar
to those formed by DnaA (Komori et al., 1999). The plasmid and chromosomal origins exhibit similar positioning
of the AT-rich region in respect to the binding sites of a
replication initiator. The iterons in plasmid origins are
clustered similarly as the DnaA-boxes in chromosomal
origins, on one side of an AT-rich DUE (Fig. 1 and text
above). The open complex formation at the plasmid origins is induced by Rep protein binding to the iterons;
however, some plasmids still require DnaA to stabilize or
enhance the origin unwinding (Kawasaki et al., 1996;
Konieczny et al., 1997; Park et al., 1998; Kruger et al.,
2001). The molecular basis for DnaA contribution to plasmid origin opening must be different comparing to mechanisms proposed for the chromosomal replication
initiation by DnaA (see above). The melting at a plasmid
AT-rich region is ATP-independent and no ATP-DnaAbox motifs are present there. It must be pointed out that
the DnaA interaction with putative DnaA-boxes within
the DUE element of RK2 plasmid was postulated (Gaylo
et al., 1987; Konieczny et al., 1997). Moreover, it could
not be excluded that during the opening reaction the Rep
proteins could interact with the 13-mer sequences within
the AT-rich region. Both in vivo and in vitro tests revealed
that specific alterations within the 13-mer sequences result
in inhibition of the RK2 oriV opening and consequently
in the lack of origin activity (Kowalczyk et al., 2005;
Rajewska et al., 2008). As the introduced alterations did
not change the internal stability pattern, these results indicate the possibility of specific protein–DNA interactions
within the AT-rich region of plasmid origins.
AT-rich region repeated sequences – helicase
loading and replication complex assembly
The AT-rich region provides structural scaffold for the
assembling of a replication complex. Regardless of a replicon analyzed, the formation of the complex involves helicase loading and subsequent assembly of primase and
ª 2011 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
M. Rajewska et al.
replicative polymerase. Those enzymes specifically bind
the ssDNA (Wickner, 1977; Rowen & Kornberg, 1978;
LeBowitz & McMacken, 1986; Kelman & O’Donnell,
1995; Jezewska et al., 1996) that is created within the
opened DUE. It is not clear if and how the nucleotide
sequence of the opened region affects the formation of a
replication complex. The E. coli DnaB helicase loading at
oriC requires both the helicase interaction with DnaA
(Marszalek & Kaguni, 1994; Sutton et al., 1998; Seitz
et al., 2000) and the involvement of an accessory ATPase,
namely DnaC, protein (Wickner & Hurwitz, 1975; Lanka
& Schuster, 1983; Davey et al., 2002). Both DnaA and
DnaC bind ssDNA (Learn et al., 1997; Messer, 2002).
ATP stimulates ssDNA binding by E. coli DnaC, leading
to the expansion of the ssDNA bubble at the origin (Davey et al., 2002). Cryptic single-stranded-DNA binding
activities of the E. coli DnaC, as well as phage lambda P
replication initiation protein, facilitates the transfer of the
E. coli DnaB helicase onto the ssDNA (Learn et al.,
1997). The determination of the structure of the ATPase
region of the DnaC from A. aeolicus demonstrates that it
is a close paralogue of the DnaA and forms a helical
assembly similar to the quaternary state adopted by ATPbound DnaA (Mott et al., 2008). Those findings implicate
that DnaC is a molecular adaptor that uses ATP-activated
DnaA as a docking site for regulating the recruitment and
correct spatial deposition of the DnaB helicase onto the
ssDNA of the opened DUE element. Two E. coli DnaB
hexamers are loaded onto the opposing strands of the
open origin structure (Fang et al., 1999). After the helicase moves approximately 65 nucleotides, the primase
synthesizes primers and two molecules of the DNA polymerase III holoenzyme are assembled on the ssDNA templates (Fang et al., 1999). SSB protein was also indicated
as a factor important for the formation and functionality
of the replication complex at the ssDNA (Meyer & Laine,
1990; Kelman et al., 1998).
The helicase loading at plasmid origins requires DnaA,
DnaC and plasmid-encoded replication initiation protein
(Konieczny & Helinski, 1997; Lu et al., 1998; Datta et al.,
1999; Pacek et al., 2001; Weigel & Seitz, 2002; Jiang et al.,
2003); however, in vitro tests indicate that in some cases
the recruitment and loading of the bacterial helicase on a
plasmid origin does not require DnaC-like ATPase (Caspi
et al., 2001; Jiang et al., 2003; Konieczny, 2003). The
DnaA and DnaC interactions with the ssDNA within the
open AT-rich region are demonstrated for chromosomal
origins (see paragraph above). It is very likely that similar
complexes, including also the plasmid Rep proteins, could
be essential for the helicase loading at plasmid origins.
During the RK2 plasmid replication initiation, the E. coli
DnaB helicase complex consisting of DnaA and DnaC
proteins was shown at dsDNA DnaA-box repeats located
FEMS Microbiol Rev 36 (2012) 408–434
425
AT-rich region and the repeated sequences in DNA replication
outside the AT-rich region of the plasmid origin (Konieczny & Helinski, 1997; Pacek et al., 2001). Specific alterations within the 13-mer sequences, which do no affect
origin opening, significantly disturb the E. coli DnaB helicase translocation from the DnaA-boxes to the RK2
AT-rich region (Rajewska et al., 2008). In case of five
mutants, this process showed that the helicase loading at
the unwound single-stranded AT-rich region depends
strictly on the activity of the plasmid’s Rep protein and
the sequence of the 13-mer repeats (Rajewska et al., 2008).
This adds to the notion of the AT-rich region being
important to replication initiation not only due to its low
internal stability. However, whether it is the sequence itself
or the sequence-dependent formation of secondary structures at the unwound origin still remains ambiguous. A
selective activation by stretches of thymine residues of the
Mcm4/6/7 helicase complex has been reported and proposed to be a determinant for the selection of DNA replication initiation sites in mammalian genomes (You et al.,
2003). Experiments on the E. coli DnaB demonstrate that
DnaA can load helicase at a DnaA-box hairpin structure
(Carr & Kaguni, 2001). The potential formation of secondary structures within the open region of origin
(Pearson et al., 1996; Rampakakis et al., 2010) may affect
the nucleoprotein interactions.
AT-rich region repeated sequences and
replication regulation
The AT-rich part of a replication origin containing 13mer repeats and sequences overlapping them, including
GATC sites, ATP-DnaA boxes, and other more or less
specified motifs for binding various regulatory proteins
(see paragraphs above) could be considered as a major
region for controlling the DNA replication initiation. This
control is achieved mainly by managing the unwinding of
an AT-rich region. The binding of the regulatory proteins
changes the DNA architecture or prevents the formation
of specific nucleoprotein complexes at the DUE. That
results in the stimulation or inhibition of origin opening.
The interactions of ATP-DnaA with specific motifs (ATPDnaA-box-like sequences) localized within the AT-rich
part of origin is critical for its opening, whereas the binding of the SeqA, IciA, ArcA, HobH, or CspD proteins to
the AT-rich sequences inhibits the formation of the open
complex and therefore the DNA replication initiation. In
contrast, the DiaA protein directly stimulates the formation of ATP-DnaA multimers on oriC and ensures timely
replication. After the melting of DUE, the interactions of
Hda and Pol III sliding clamp with ATP-DnaA and
ssDNA within the open region inactivate DnaA protein.
This prevents an immediate ATP-dependent activity of
DnaA and as a result an uncontrolled origin opening.
FEMS Microbiol Rev 36 (2012) 408–434
The replication initiation could possibly be also regulated
by controlling the formation of a helicase complex at the
unwound AT-rich structure. It was recently proposed that
the DiaA dynamics is coupled with changes in the initial
complexes at the origin, leading to helicase loading (Keyamura et al., 2009). Moreover, the NAPs such as Fis,
HU, IHF affect the origin’s architecture and distribution
of the DnaA protein and, therefore, the activity of replication origin. In some replicons, the IHF-binding sites
are located within the DUE (see earlier paragraphs), indicating the relevance of the NAPs interactions within this
region. A model for the dynamic interplay between the
DNA architectural proteins and DnaA during replication
initiation has been proposed (Mott & Berger, 2007). The
AT-rich region of a replication origin accommodates
multiple protein interactions and conformations. The formation of oligomeric structures composed of replication
initiators and architectural proteins as well as specific
interactions of regulatory factors seem to be the key factors for origin activities. A specific nucleotide sequence of
DUE element is essential and provides specificity of
nucleoprotein interactions during the process of replication initiation and its control.
Summary
A common feature of many bacterial origins of replication is the presence of a region rich in adenine and thymine residues. These AT-rich regions are the sites where
the processes of DNA unwinding and replication start.
They are usually characterized by low thermodynamic stability in comparison with the overall origin stability and
contain repeated sequences of various lengths that are
crucial for the proper functioning of the replicons. The
presence of the repeated motifs causes fluctuations, characteristic ‘intervals’ and differences among the minima of
free energy, which can be observed when analyzing the
thermodynamic stability patterns of particular origins.
Patterns of the internal stability may also be related to
the helical periodicity of some of the repeats within the
AT-rich regions. In the bacterial chromosomal origins of
replication, the AT-rich regions are usually located at one
side of a cluster of DnaA-box sequences. In plasmids,
they are usually followed by one or two DnaA-boxes and
precede the binding sites for plasmid initiator protein.
Such arrangement of the motifs seems to be of great
importance for the efficient functioning of replication origins. It is possible that proper organization of the motifs
might be connected with the function of initiator proteins
and the mechanism of replication initiation at a particular origin. The AT-rich regions are of different sizes,
depending on the origin; however, what seems to be
common for them is the size of the initial DNA opening
ª 2011 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
M. Rajewska et al.
426
at DUE, which is very rarely longer than 50 bp and no
shorter than 20 bp. This extent of origin opening seems
to be sufficient for the assembly of the prereplication
complex at the unwound site.The great majority of the
AT-rich regions of prokaryotic origins contain repeats of
various lengths. They appear to be absolutely critical for
the origin functioning and the formation of protein complexes during replication initiation events. In the bacterial
origins, the repeats within the AT-rich regions are usually
13 nucleotides long and possess high sequence similarity
with consensus determined for E. coli oriC 13-mers. They
also contain a distinctive core consisting mostly of adenines and thymines at the right-hand side of each repeat.
Most often, there are three direct repeats in the region
that play a direct role in the open complex formation at
the origin during the replication initiation. In plasmids,
the repeats vary both in length and sequence and establishing a general consensus for them is rather difficult.
However, the length, number, and spacing between the
repeats are individual for each plasmid origin and the
specific sequence of each is substantial for origin functioning. Biochemical analyses of replication initiation at
different origins revealed that the sequence or, possibly,
the secondary DNA structure resulting from it is absolutely critical for the replicating activity of a replicon.
However, whether it is the linear or secondary structure
of DNA in the AT-rich region, which is crucial for origin’s activity and multiprotein interactions in this region,
still requires investigation. Within the repeats of the chromosomal and plasmid AT-rich regions overalapping
motifs for binding proteins engaged in replication initiation and its regulation were also identified. However, the
exact role of the particular motifs is also being investigated. It is highly interesting whether the multiprotein
interactions within the AT-rich region are competitive or
maybe the accumulation of motifs for protein interactions
plays a role at different stages of replication and allows
separation of particular events of the process. The analysis
of the nucleoprotein complexes formed at the AT-rich
region of replication origin will be crucial for further
understanding of the regulation and molecular mechanisms involved in the initial origin opening and subsequent steps of replication initiation.
Acknowledgements
This work was supported by grant N N301 295837 from
Polish Ministry of Science and Higher Education. We
thank Foundation for Polish Science for support under
TEAM (for I.K.) and START (for M.R.) programs. M.R.
was also financed from European Social Fund in the
framework of the Program of implementing the elements
of modern education forms at the University of Gdansk.
ª 2011 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
References
Abdurashidova G, Deganuto M, Klima R, Riva S, Biamonti G,
Giacca M & Falaschi A (2000) Start sites of bidirectional
DNA synthesis at the human lamin B2 origin. Science 287:
2023–2026.
Abeles AL (1986) P1 plasmid replication. Purification and
DNA-binding activity of the replication protein RepA. J Biol
Chem 261: 3548–3555.
Abeles AL, Snyder KM & Chattoraj DK (1984) P1 plasmid
replication: replicon structure. J Mol Biol 173: 307–324.
Abeles AL, Reaves LD & Austin SJ (1990) A single DnaA box
is sufficient for initiation from the P1 plasmid origin.
J Bacteriol 172: 4386–4391.
Abeles A, Brendler T & Austin S (1993) Evidence of two
levels of control of P1 oriR and host oriC replication
origins by DNA adenine methylation. J Bacteriol 175:
7801–7807.
Abhyankar MM, Zzaman S & Bastia D (2003) Reconstitution
of R6K DNA replication in vitro using 22 purified proteins.
J Biol Chem 278: 45476–45484.
Asai T, Takanami M & Imai M (1990) The AT richness and
gid transcription determine the left border of the replication
origin of the E. coli chromosome. EMBO J 9: 4065–4072.
Bell SP & Dutta A (2002) DNA replication in eukaryotic cells.
Annu Rev Biochem 71: 333–374.
Bernander R, Dasgupta S & Nordstrom K (1991) The E. coli
cell cycle and the plasmid R1 replication cycle in the
absence of the DnaA protein. Cell 64: 1145–1153.
Bernander R, Krabbe M & Nordstrom K (1992) Mapping of
the in vivo start site for leading strand DNA synthesis in
plasmid R1. EMBO J 11: 4481–4487.
Berquist BR & DasSarma S (2003) An archaeal chromosomal
autonomously replicating sequence element from an
extreme halophile, Halobacterium sp. strain NRC-1.
J Bacteriol 185: 5959–5966.
Bielinsky AK & Gerbi SA (1998) Discrete start sites for DNA
synthesis in the yeast ARS1 origin. Science 279: 95–98.
Bielinsky AK & Gerbi SA (1999) Chromosomal ARS1 has a
single leading strand start site. Mol Cell 3: 477–486.
Bielinsky AK & Gerbi SA (2001) Where it all starts: eukaryotic
origins of DNA replication. J Cell Sci 114: 643–651.
Blaesing F, Weigel C, Welzeck M & Messer W (2000) Analysis
of the DNA-binding domain of Escherichia coli DnaA
protein. Mol Microbiol 36: 557–569.
Bowers LM, Kruger R & Filutowicz M (2007) Mechanism of
origin activation by monomers of R6K-encoded pi protein.
J Mol Biol 368: 928–938.
Bramhill D & Kornberg A (1988a) A model for initiation at
origins of DNA replication. Cell 54: 915–918.
Bramhill D & Kornberg A (1988b) Duplex opening by DnaA
protein at novel sequences in initiation of replication at the
origin of the E. coli chromosome. Cell 52: 743–755.
Breier AM, Chatterji S & Cozzarelli NR (2004) Prediction of
Saccharomyces cerevisiae replication origins. Genome Biol 5:
R22.
FEMS Microbiol Rev 36 (2012) 408–434
AT-rich region and the repeated sequences in DNA replication
Brendler T, Abeles A & Austin S (1991a) Critical sequences in
the core of the P1 plasmid replication origin. J Bacteriol
173: 3935–3942.
Brendler TG, Abeles AL, Reaves LD & Austin SJ (1991b)
Unique sequence requirements for the P1 plasmid
replication origin. Res Microbiol 142: 209–216.
Brendler T, Abeles A & Austin S (1995) A protein that binds
to the P1 origin core and the oriC 13mer region in a
methylation-specific fashion is the product of the host seqA
gene. EMBO J 14: 4083–4089.
Camara JE, Breier AM, Brendler T, Austin S, Cozzarelli NR &
Crooke E (2005) Hda inactivation of DnaA is the
predominant mechanism preventing hyperinitiation of
Escherichia coli DNA replication. EMBO Rep 6: 736–741.
Carr KM & Kaguni JM (2001) Stoichiometry of DnaA and
DnaB protein in initiation at the Escherichia coli
chromosomal origin. J Biol Chem 276: 44919–44925.
Caspi R, Pacek M, Consiglieri G, Helinski DR, Toukdarian AE
& Konieczny I (2001) A broad host range replicon with
different requirements for replication initiation in three
bacterial species. EMBO J 20: 3262–3271.
Chattoraj DK, Snyder KM & Abeles AL (1985) P1 plasmid
replication: multiple functions of RepA protein at the
origin. P Natl Acad Sci USA 82: 2588–2592.
Chew DS, Leung MY & Choi KP (2007) AT excursion: a new
approach to predict replication origins in viral genomes by
locating AT-rich regions. BMC Bioinformatics 8: 163.
Chiang CS, Xu YC & Bremer H (1991) Role of DnaA protein
during replication of plasmid pBR322 in Escherichia coli.
Mol Gen Genet 225: 435–442.
Churchward G, Linder P & Caro L (1983) The nucleotide
sequence of replication and maintenance functions encoded
by plasmid pSC101. Nucleic Acids Res 11: 5645–5659.
Coker JA, DasSarma P, Capes M et al. (2009) Multiple
replication origins of Halobacterium sp. strain NRC-1:
properties of the conserved orc7-dependent oriC1.
J Bacteriol 191: 5253–5261.
Crosa JH, Luttropp LK & Falkow S (1978) Molecular cloning
of replication and incompatibility regions from the
R-plasmid R6K. J Mol Biol 124: 443–468.
Danbara H, Timmis JK, Lurz R & Timmis KN (1980) Plasmid
replication functions: two distinct segments of plasmid R1,
RepA and RepD, express incompatibility and are capable of
autonomous replication. J Bacteriol 144: 1126–1138.
Datta HJ, Khatri GS & Bastia D (1999) Mechanism of
recruitment of DnaB helicase to the replication origin of the
plasmid pSC101. P Natl Acad Sci USA 96: 73–78.
Davey MJ, Fang L, McInerney P, Georgescu RE & O’Donnell
M (2002) The DnaC helicase loader is a dual ATP/ADP
switch protein. EMBO J 17: 3148–3159.
del Solar G, Giraldo R, Ruiz-Echevarria MJ, Espinosa M &
Diaz-Orejas R (1998) Replication and control of circular
bacterial plasmids. Microbiol Mol Biol Rev 62: 434–464.
Dellis S & Filutowicz M (1991) Integration host factor of
Escherichia coli reverses the inhibition of R6K plasmid
replication by pi initiator protein. J Bacteriol 173: 1279–1286.
FEMS Microbiol Rev 36 (2012) 408–434
427
Dellis S, Feng J & Filutowicz M (1996) Replication of plasmid
R6K gamma origin in vivo and in vitro: dependence on IHF
binding to the ihf1 site. J Mol Biol 257: 550–560.
DePamphilis ML (1999) Replication origins in metazoan
chromosomes: fact or fiction? Bioessays 21: 5–16.
Diaz R & Staudenbauer WL (1982) Origin and direction of
mini-R1 plasmid DNA replication in cell extracts of
Escherichia coli. J Bacteriol 150: 1077–1084.
Diaz-Lopez T, Lages-Gonzalo M, Serrano-Lopez A, Alfonso
C, Rivas G, Diaz-Orejas R & Giraldo R (2003)
Structural changes in RepA, a plasmid replication
initiator, upon binding to origin DNA. J Biol Chem 278:
18606–18616.
Dijkwel PA & Hamlin JL (1995) The Chinese hamster
dihydrofolate reductase origin consists of multiple potential
nascent-strand start sites. Mol Cell Biol 15: 3023–3031.
Dodson M, McMacken R & Echols H (1989) Specialized
nucleoprotein structures at the origin of replication of
bacteriophage lambda. Protein association and
disassociation reactions responsible for localized initiation of
replication. J Biol Chem 264: 10719–10725.
Doran KS, Konieczny I & Helinski DR (1998) Replication
origin of the broad host range plasmid RK2. Positioning of
various motifs is critical for initiation of replication. J Biol
Chem 273: 8447–8453.
Doran KS, Helinski DR & Konieczny I (1999a) A critical
DnaA box directs the cooperative binding of the Escherichia
coli DnaA protein to the plasmid RK2 replication origin.
J Biol Chem 274: 17918–17923.
Doran KS, Helinski DR & Konieczny I (1999b) Hostdependent requirement for specific DnaA boxes for plasmid
RK2 replication. Mol Microbiol 33: 490–498.
Duderstadt KE, Mott ML, Crisona NJ, Chuang K, Yang H &
Berger JM (2010) Origin remodeling and opening in
bacteria rely on distinct assembly states of the DnaA
initiator. J Biol Chem 285: 28229–28239.
Egan ES & Waldor MK (2003) Distinct replication
requirements for the two Vibrio cholerae chromosomes. Cell
114: 521–530.
Eichenlaub R, Figurski D & Helinski DR (1977) Bidirection
replication from a unique origin in a mini-F plasmid.
P Natl Acad Sci USA 74: 1138–1141.
Erzberger JP, Pirruccello MM & Berger JM (2002) The
structure of bacterial DnaA: implications for general
mechanisms underlying DNA replication initiation. EMBO J
21: 4763–4773.
Fang L, Davey MJ & O’Donnell M (1999) Replisome assembly at
oriC, the replication origin of E. coli, reveals an explanation
for initiation sites outside an origin. Mol Cell 4: 541–553.
Fekete RA, Venkova-Canova T, Park K & Chattoraj DK (2006)
IHF-dependent activation of P1 plasmid origin by dnaA.
Mol Microbiol 62: 1739–1751.
Filutowicz M & Appelt K (1988) The integration host factor of
Escherichia coli binds to multiple sites at plasmid R6K
gamma origin and is essential for replication. Nucleic Acids
Res 16: 3829–3843.
ª 2011 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
428
Frank AC & Lobry JR (2000) Oriloc: prediction of replication
boundaries in unannotated bacterial chromosomes.
Bioinformatics 16: 560–561.
Freeman JM, Plasterer TN, Smith TF & Mohr SC (1998)
Patterns of genome organization in bacteria. Science 279:
1827.
Fukuoka T, Moriya S, Yoshikawa H & Ogasawara N (1990)
Purification and characterization of an initiation protein for
chromosomal replication, DnaA, in Bacillus subtilis.
J Biochem 107: 732–739.
Fuller RS, Funnell BE & Kornberg A (1984) The dnaA protein
complex with the E. coli chromosomal replication origin
(oriC) and other DNA sites. Cell 38: 889–900.
Gajiwala KS & Burley SK (2000) Winged helix proteins. Curr
Opin Struct Biol 10: 110–116.
Gao F & Zhang CT (2007) DoriC: a database of oriC regions
in bacterial genomes. Bioinformatics 23: 1866–1867.
Gao F & Zhang CT (2008) Ori-Finder: a web-based system for
finding oriCs in unannotated bacterial genomes. BMC
Bioinformatics 9: 79.
Gaylo PJ, Turjman N & Bastia D (1987) DnaA protein is
required for replication of the minimal replicon of the
broad-host-range plasmid RK2 in Escherichia coli. J Bacteriol
169: 4703–4709.
Geier GE & Modrich P (1979) Recognition sequence of the
dam methylase of Escherichia coli K12 and mode of cleavage
of Dpn I endonuclease. J Biol Chem 254: 1408–1413.
Gerbi SA & Bielinsky AK (1997) Replication initiation point
mapping. Methods 13: 271–280.
Gille H & Messer W (1991) Localized DNA melting and
structural pertubations in the origin of replication, oriC, of
Escherichia coli in vitro and in vivo. EMBO J 10: 1579–1584.
Gille H, Egan JB, Roth A & Messer W (1991) The FIS protein
binds and bends the origin of chromosomal DNA
replication, oriC, of Escherichia coli. Nucleic Acids Res 19:
4167–4172.
Giraldo R (2003) Common domains in the initiators of DNA
replication in Bacteria, Archaea and Eukarya: combined
structural, functional and phylogenetic perspectives. FEMS
Microbiol Rev 26: 533–554.
Giraldo R & Diaz R (1992) Differential binding of wild-type
and a mutant RepA protein to oriR sequence suggests a
model for the initiation of plasmid R1 replication. J Mol
Biol 228: 787–802.
Giraldo R & Fernandez-Tresguerres ME (2004) Twenty years
of the pPS10 replicon: insights on the molecular mechanism
for the activation of DNA replication in iteron-containing
bacterial plasmids. Plasmid 52: 69–83.
Gomez M & Antequera F (1999) Organization of DNA
replication origins in the fission yeast genome. EMBO J 18:
5683–5690.
Gonzalez-Soltero R, Botello E & Jimenez-Sanchez A (2006)
Initiation of heat-induced replication requires DnaA and the
L-13-mer of oriC. J Bacteriol 188: 8294–8298.
Graumann P & Marahiel MA (1994) The major cold shock
protein of Bacillus subtilis CspB binds with high affinity to
ª 2011 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
M. Rajewska et al.
the ATTGG- and CCAAT sequences in single stranded
oligonucleotides. FEBS Lett 338: 157–160.
Graumann P, Wendrich TM, Weber MH, Schroder K &
Marahiel MA (1997) A family of cold shock proteins in
Bacillus subtilis is essential for cellular growth and for
efficient protein synthesis at optimal and low temperatures.
Mol Microbiol 25: 741–756.
Grigoriev A (1998) Analyzing genomes with cumulative skew
diagrams. Nucleic Acids Res 26: 2286–2290.
Grimwade JE, Torgue JJ, McGarry KC, Rozgaja T, Enloe ST &
Leonard AC (2007) Mutational analysis reveals Escherichia
coli oriC interacts with both DnaA-ATP and DnaA-ADP
during pre-RC assembly. Mol Microbiol 66: 428–439.
Guarne A, Zhao Q, Ghirlando R & Yang W (2002) Insights
into negative modulation of E. coli replication initiation
from the structure of SeqA-hemimethylated DNA complex.
Nat Struct Biol 9: 839–843.
Guarne A, Brendler T, Zhao Q, Ghirlando R, Austin S & Yang
W (2005) Crystal structure of a SeqA-N filament:
implications for DNA replication and chromosome
organization. EMBO J 24: 1502–1511.
Hamlin JL & Dijkwel PA (1995) On the nature of replication
origins in higher eukaryotes. Curr Opin Genet Dev 5: 153–
161.
Han JS, Kang S, Lee H, Kim HK & Hwang DS (2003)
Sequential binding of SeqA to paired hemi-methylated
GATC sequences mediates formation of higher order
complexes. J Biol Chem 278: 34983–34989.
Hansen EB & Yarmolinsky MB (1986) Host participation in
plasmid maintenance: dependence upon dnaA of replicons
derived from P1 and F. P Natl Acad Sci USA 83: 4423–4427.
Hansen FG, Christensen BB, Nielsen CB & Atlung T (2006)
Insights into the quality of DnaA boxes and their
cooperativity. J Mol Biol 355: 85–95.
Herrick J, Kern R, Guha S, Landoulsi A, Fayet O, Malki A &
Kohiyama M (1994) Parental strand recognition of the
DNA replication origin by the outer membrane in
Escherichia coli. EMBO J 13: 4695–4703.
Hsu J, Bramhill D & Thompson CM (1994) Open
complex formation by DnaA initiator protein at the
Escherichia coli chromosomal origin requires the 13-mer
precisely spaced relative to the 9-mers. Mol Microbiol 11:
903–911.
Hwang DS & Kornberg A (1990) A novel protein binds a key
origin sequence to block replication of an E. coli
minichromosome. Cell 63: 325–331.
Hwang DS & Kornberg A (1992a) Opening of the replication
origin of Escherichia coli by DnaA protein with protein HU
or IHF. J Biol Chem 267: 23083–23086.
Hwang DS & Kornberg A (1992b) Opposed actions of
regulatory proteins, DnaA and IciA, in opening the
replication origin of Escherichia coli. J Biol Chem 267:
23087–23091.
Inselburg J (1974) Replication of colicin E1 plasmid DNA in
minicells from a unique replication initiation site. P Natl
Acad Sci USA 71: 2256–2259.
FEMS Microbiol Rev 36 (2012) 408–434
AT-rich region and the repeated sequences in DNA replication
Ishiai M, Wada C, Kawasaki Y & Yura T (1994) Replication
initiator protein RepE of mini-F plasmid: functional
differentiation between monomers (initiator) and dimers
(autogenous repressor). P Natl Acad Sci USA 91: 3839–3843.
Ishida T, Akimitsu N, Kashioka T et al. (2004) DiaA, a novel
DnaA-binding protein, ensures the timely initiation of
Escherichia coli chromosome replication. J Biol Chem 279:
45546–45555.
Jacob F & Brenner S (1963) [On the regulation of DNA
synthesis in bacteria: the hypothesis of the replicon].
CR Hebd Seances Acad Sci 256: 298–300.
Jakimowicz D, Majka J & Messer W et al. (1998) Structural
elements of the Streptomyces oriC region and their
interactions with the DnaA protein. Microbiology 144 (Pt 5):
1281–1290.
Jezewska MJ, Kim US & Bujalowski W (1996) Binding of
Escherichia coli primary replicative helicase DnaB protein to
single-stranded DNA. Long-range allosteric conformational
changes within the protein hexamer. Biochemistry 35: 2129–
2145.
Jiang W, Hou Y & Inouye M (1997) CspA, the major coldshock protein of Escherichia coli, is an RNA chaperone.
J Biol Chem 272: 196–202.
Jiang Y, Pacek M, Helinski DR, Konieczny I & Toukdarian A
(2003) A multifunctional plasmid-encoded replication
initiation protein both recruits and positions an active
helicase at the replication origin. P Natl Acad Sci USA 100:
8692–8697.
Kang S, Lee H, Han JS & Hwang DS (1999) Interaction of
SeqA and Dam methylase on the hemimethylated origin of
Escherichia coli chromosomal DNA replication. J Biol Chem
274: 11463–11468.
Kang S, Han JS, Kim KP, Yang HY, Lee KY, Hong CB &
Hwang DS (2005) Dimeric configuration of SeqA protein
bound to a pair of hemi-methylated GATC sequences.
Nucleic Acids Res 33: 1524–1531.
Kano Y, Ogawa T, Ogura T, Hiraga S, Okazaki T & Imamoto
F (1991) Participation of the histone-like protein HU and of
IHF in minichromosomal maintenance in Escherichia coli.
Gene 103: 25–30.
Katayama T, Kubota T, Kurokawa K, Crooke E & Sekimizu K
(1998) The initiator function of DnaA protein is negatively
regulated by the sliding clamp of the E. coli chromosomal
replicase. Cell 94: 61–71.
Katayama T, Ozaki S, Keyamura K & Fujimitsu K (2010)
Regulation of the replication cycle: conserved and diverse
regulatory systems for DnaA and oriC. Nat Rev Microbiol 8:
163–170.
Kawasaki Y, Matsunaga F, Kano Y, Yura T & Wada C (1996)
The localized melting of mini-F origin by the combined
action of the mini-F initiator protein (RepE) and HU and
DnaA of Escherichia coli. Mol Gen Genet 253: 42–49.
Kelley WL & Bastia D (1992) Activation in vivo of the
minimal replication origin beta of plasmid R6K requires a
small target sequence essential for DNA looping. New Biol
4: 569–580.
FEMS Microbiol Rev 36 (2012) 408–434
429
Kelley WL, Patel I & Bastia D (1992) Structural and functional
analysis of a replication enhancer: separation of the
enhancer activity from origin function by mutational
dissection of the replication origin gamma of plasmid R6K.
P Natl Acad Sci USA 89: 5078–5082.
Kelman Z & O’Donnell M (1995) DNA polymerase III
holoenzyme: structure and function of a chromosomal
replicating machine. Annu Rev Biochem 64: 171–200.
Kelman Z, Yuzhakov A, Andjelkovic J & O’Donnell M (1998)
Devoted to the lagging strand-the subunit of DNA
polymerase III holoenzyme contacts SSB to promote
processive elongation and sliding clamp assembly. EMBO J
17: 2436–2449.
Keyamura K, Abe Y, Higashi M, Ueda T & Katayama T (2009)
DiaA dynamics are coupled with changes in initial origin
complexes leading to helicase loading. J Biol Chem 284:
25038–25050.
Kim MS, Bae SH, Yun SH et al. (2005) Cnu, a novel oriCbinding protein of Escherichia coli. J Bacteriol 187: 6998–
7008.
Kline BC, Kogoma T, Tam JE & Shields MS (1986)
Requirement of the Escherichia coli dnaA gene product for
plasmid F maintenance. J Bacteriol 168: 440–443.
Koch B, Ma X & Lobner-Olesen A (2010) Replication of
Vibrio cholerae chromosome I in Escherichia coli:
dependence on dam methylation. J Bacteriol 192: 3903–
3914.
Kolter R & Helinski DR (1978) Construction of plasmid R6K
derivatives in vitro: characterization of the R6K replication
region. Plasmid 1: 571–580.
Kolter R & Helinski DR (1982) Plasmid R6K DNA replication.
II. Direct nucleotide sequence repeats are required for an
active gamma-origin. J Mol Biol 161: 45–56.
Komori H, Matsunaga F, Higuchi Y, Ishiai M, Wada C & Miki
K (1999) Crystal structure of a prokaryotic replication
initiator protein bound to DNA at 2.6 A resolution. EMBO
J 18: 4597–4607.
Konieczny I (2003) Strategies for helicase recruitment and
loading in bacteria. EMBO Rep 4: 37–41.
Konieczny I & Helinski DR (1997) Helicase delivery and
activation by DnaA and TrfA proteins during the initiation
of replication of the broad host range plasmid RK2. J Biol
Chem 272: 33312–33318.
Konieczny I, Doran KS, Helinski DR & Blasina A (1997) Role
of TrfA and DnaA proteins in origin opening during
initiation of DNA replication of the broad host range
plasmid RK2. J Biol Chem 272: 20173–20178.
Kornberg A & Baker T (1992) DNA Replication. W.H.
Freeman & Co., New York.
Kowalczyk L, Rajewska M & Konieczny I (2005) Positioning
and the specific sequence of each 13-mer motif are critical
for activity of the plasmid RK2 replication origin. Mol
Microbiol 57: 1439–1449.
Kowalski D & Eddy MJ (1989) The DNA unwinding element:
a novel, cis-acting component that facilitates opening of the
Escherichia coli replication origin. EMBO J 8: 4335–4344.
ª 2011 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
430
Krause M, Ruckert B, Lurz R & Messer W (1997) Complexes
at the replication origin of Bacillus subtilis with homologous
and heterologous DnaA protein. J Mol Biol 274: 365–380.
Kruger R & Filutowicz M (2000) Dimers of pi protein bind
the A+T-rich region of the R6K gamma origin near the
leading-strand synthesis start sites: regulatory implications.
J Bacteriol 182: 2461–2467.
Kruger R & Filutowicz M (2003) pi protein- and ATPdependent transitions from ‘closed’ to ‘open’ complexes at
the gamma ori of plasmid R6K. Nucleic Acids Res 31: 5993–
6003.
Kruger R, Konieczny I & Filutowicz M (2001) Monomer/dimer
ratios of replication protein modulate the DNA strandopening in a replication origin. J Mol Biol 306: 945–955.
Kunnimalaiyaan S, Kruger R, Ross W, Rakowski SA &
Filutowicz M (2004) Binding modes of the initiator and
inhibitor forms of the replication protein pi to the gamma
ori iteron of plasmid R6K. J Biol Chem 279: 41058–41066.
Kurz M, Dalrymple B, Wijffels G & Kongsuwan K (2004)
Interaction of the sliding clamp beta-subunit and Hda, a
DnaA-related protein. J Bacteriol 186: 3508–3515.
Kuzminov A, Schabtach E & Stahl FW (1997) Study of
plasmid replication in Escherichia coli with a combination of
2D gel electrophoresis and electron microscopy. J Mol Biol
268: 1–7.
Lanka E & Schuster H (1983) The dnaC protein of Escherichia
coli. Purification, physical properties and interaction with
dnaB protein. Nucleic Acids Res 11: 987–997.
Learn BA, Um S-J, Li H & McMacken R (1997) Cryptic
single-stranded-DNA binding activities of the phage lambda
P and Escherichia coli DnaC replication initiation proteins
facilitate the transfer of E. coli DnaB helicase onto DNA.
P Natl Acad Sci USA 94: 1154–1159.
LeBowitz JH & McMacken R (1986) The Escherichia coli dnaB
replication protein is a DNA helicase. J Biol Chem 261:
4738–4748.
Lee H, Kang S, Bae SH, Choi BS & Hwang DS (2001a) SeqA
protein aggregation is necessary for SeqA function. J Biol
Chem 276: 34600–34606.
Lee YS, Han JS, Jeon Y & Hwang DS (2001b) The arc twocomponent signal transduction system inhibits in vitro
Escherichia coli chromosomal initiation. J Biol Chem 276:
9917–9923.
Leonard AC & Grimwade JE (2005) Building a bacterial
orisome: emergence of new regulatory features
for replication origin unwinding. Mol Microbiol 55: 978–
985.
Levchenko I & Filutowicz M (1996) Initiator protein pi can
bind independently to two domains of the gamma origin
core of plasmid R6K: the direct repeats and the A+T-rich
segment. Nucleic Acids Res 24: 1936–1942.
Lobry JR (1996a) Asymmetric substitution patterns in the two
DNA strands of bacteria. Mol Biol Evol 13: 660–665.
Lobry JR (1996b) A simple vectorial representation of DNA
sequences for the detection of replication origins in bacteria.
Biochimie 78: 323–326.
ª 2011 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
M. Rajewska et al.
Lopez P, Philippe H, Myllykallio H & Forterre P (1999)
Identification of putative chromosomal origins of
replication in Archaea. Mol Microbiol 32: 883–886.
Lopez P, Forterre P, le Guyader H & Philippe H (2000)
Origin of replication of Thermotoga maritima. Trends Genet
16: 59–60.
Lu M, Campbell JL, Boye E & Kleckner N (1994) SeqA: a
negative modulator of replication initiation in E. coli. Cell
77: 413–426.
Lu Y, Datta HJ & Bastia D (1998) Mechanistic studies of
initiator-initiator interaction and replication initiation.
EMBO J 17: 5192–5200.
Lundgren M, Andersson A, Chen L, Nilsson P & Bernander R
(2004) Three replication origins in Sulfolobus species:
synchronous initiation of chromosome replication and
asynchronous termination. P Natl Acad Sci USA 101: 7046–
7051.
MacAllister TW, Kelley WL, Miron A, Stenzel TT & Bastia D
(1991) Replication of plasmid R6K origin gamma in vitro.
Dependence on dual initiator proteins and inhibition by
transcription. J Biol Chem 266: 16056–16062.
Mackiewicz P, Gierlik A, Kowalczuk M, Dudek MR & Cebrat
S (1999a) How does replication-associated mutational
pressure influence amino acid composition of proteins?
Genome Res 9: 409–416.
Mackiewicz P, Gierlik A, Kowalczuk M, Dudek MR &
Cebrat S (1999b) Asymmetry of nucleotide composition
of prokaryotic chromosomes. J Appl Genet 40: 1–14.
Mackiewicz P, Zakrzewska-Czerwinska J, Zawilak A, Dudek
MR & Cebrat S (2004) Where does bacterial replication
start? Rules for predicting the oriC region. Nucleic Acids Res
32: 3781–3791.
Manen D & Caro L (1991) The replication of plasmid pSC101.
Mol Microbiol 5: 233–237.
Manen D, Upegui-Gonzalez LC & Caro L (1992) Monomers and
dimers of the RepA protein in plasmid pSC101 replication:
domains in RepA. P Natl Acad Sci USA 89: 8923–8927.
Marczynski GT & Shapiro L (1992) Cell-cycle control of a
cloned chromosomal origin of replication from Caulobacter
crescentus. J Mol Biol 226: 959–977.
Margolin W & Long SR (1993) Isolation and characterization
of a DNA replication origin from the 1,700-kilobase-pair
symbiotic megaplasmid pSym-b of Rhizobium meliloti.
J Bacteriol 175: 6553–6561.
Margulies C & Kaguni JM (1996) Ordered and sequential
binding of DnaA protein to oriC, the chromosomal origin
of Escherichia coli. J Biol Chem 271: 17035–17040.
Marszalek J & Kaguni JM (1994) DnaA protein directs the
binding of DnaB protein in initiation of DNA replication in
Escherichia coli. J Biol Chem 269: 4883–4890.
Masson L & Ray DS (1988) Mechanism of autonomous
control of the Escherichia coli F plasmid: purification and
characterization of the repE gene product. Nucleic Acids Res
16: 413–424.
Matsui M, Oka A, Takanami M, Yasuda S & Hirota Y (1985)
Sites of dnaA protein-binding in the replication origin of
FEMS Microbiol Rev 36 (2012) 408–434
AT-rich region and the repeated sequences in DNA replication
the Escherichia coli K-12 chromosome. J Mol Biol 184: 529–
533.
Matsunaga F, Kawasaki Y, Ishiai M, Nishikawa K, Yura T &
Wada C (1995) DNA-binding domain of the RepE initiator
protein of mini-F plasmid: involvement of the carboxylterminal region. J Bacteriol 177: 1994–2001.
Matsunaga F, Forterre P, Ishino Y & Myllykallio H (2001)
In vivo interactions of archaeal Cdc6/Orc1 and
minichromosome maintenance proteins with the replication
origin. P Natl Acad Sci USA 98: 11152–11157.
Maurizi MR & Li CC (2001) AAA proteins: in search of a
common molecular basis. International Meeting on
Cellular Functions of AAA Proteins. EMBO Rep 2: 980–
985.
McEachern MJ, Filutowicz M & Helinski DR (1985) Mutations
in direct repeat sequences and in a conserved sequence
adjacent to the repeats result in a defective replication
origin in plasmid R6K. P Natl Acad Sci USA 82: 1480–1484.
McGarry KC, Ryan VT, Grimwade JE & Leonard AC (2004)
Two discriminatory binding sites in the Escherichia coli
replication origin are required for DNA strand opening by
initiator DnaA-ATP. P Natl Acad Sci USA 101: 2811–2816.
McLean MJ, Wolfe KH & Devine KM (1998) Base
composition skews, replication orientation, and gene
orientation in 12 prokaryote genomes. J Mol Evol 47: 691–
696.
Meijer M, Beck E, Hansen FG, Bergmans HE, Messer W,
von Meyenburg K & Schaller H (1979) Nucleotide
sequence of the origin of replication of the Escherichia
coli K-12 chromosome. P Natl Acad Sci USA 76: 580–584.
Mendelson I, Gottesman M & Oppenheim AB (1991) HU and
integration host factor function as auxiliary proteins in
cleavage of phage lambda cohesive ends by terminase.
J Bacteriol 173: 1670–1676.
Messer W (2002) The bacterial replication initiator DnaA.
DnaA and oriC, the bacterial mode to initiate DNA
replication. FEMS Microbiol Rev 26: 355–374.
Messer W, Meijer M, Bergmans HE, Hansen FG, von
Meyenburg K, Beck E & Schaller H (1979) Origin of
replication, oriC, of the Escherichia coli K12 chromosome:
nucleotide sequence. Cold Spring Harb Symp Quant Biol 43
(Pt 1): 139–145.
Messer W, Blaesing F, Jakimowicz D et al. (2001) Bacterial
replication initiator DnaA. Rules for DnaA binding and
roles of DnaA in origin unwinding and helicase loading.
Biochimie 83: 5–12.
Meyer RR & Laine PS (1990) The single-stranded DNA-binding
protein of Escherichia coli. Microbiol Rev 54: 342–380.
Miller C, Ingmer H, Thomsen LE, Skarstad K & Cohen SN
(2003) DpiA binding to the replication origin of Escherichia
coli plasmids and chromosomes destabilizes plasmid
inheritance and induces the bacterial SOS response.
J Bacteriol 185: 6025–6031.
Moriya S, Ogasawara N & Yoshikawa H (1985) Structure and
function of the region of the replication origin of the
Bacillus subtilis chromosome. III. Nucleotide sequence of
FEMS Microbiol Rev 36 (2012) 408–434
431
some 10,000 base pairs in the origin region. Nucleic Acids
Res 13: 2251–2265.
Moriya S, Atlung T, Hansen FG, Yoshikawa H & Ogasawara N
(1992) Cloning of an autonomously replicating sequence
(ars) from the Bacillus subtilis chromosome. Mol Microbiol
6: 309–315.
Moriya S, Firshein W, Yoshikawa H & Ogasawara N (1994)
Replication of a Bacillus subtilis oriC plasmid in vitro. Mol
Microbiol 12: 469–478.
Mott ML & Berger JM (2007) DNA replication initiation:
mechanisms and regulation in bacteria. Nat Rev Microbiol 5:
343–354.
Mott ML, Erzberger JP, Coons MM & Berger JM (2008)
Structural synergy and molecular crosstalk between bacterial
helicase loaders and replication initiators. Cell 135: 623–634.
Mrazek J & Karlin S (1998) Strand compositional asymmetry
in bacterial and large viral genomes. P Natl Acad Sci USA
95: 3720–3725.
Mukherjee S, Patel I & Bastia D (1985) Conformational
changes in a replication origin induced by an initiator
protein. Cell 43: 189–197.
Mukherjee S, Erickson H & Bastia D (1988) Enhancer-origin
interaction in plasmid R6K involves a DNA loop mediated
by initiator protein. Cell 52: 375–383.
Mukhopadhyay G, Carr KM, Kaguni JM & Chattoraj DK
(1993) Open-complex formation by the host initiator,
DnaA, at the origin of P1 plasmid replication. EMBO J 12:
4547–4554.
Murakami Y, Ohmori H, Yura T & Nagata T (1987)
Requirement of the Escherichia coli dnaA gene function for
ori-2-dependent mini-F plasmid replication. J Bacteriol 169:
1724–1730.
Murakami A, Sugiura S & Yamaguchi K (1997) DNA bending
of pSC101 ori induced by Rep, a replication initiator
protein and IHF. J Gen Appl Microbiol 43: 189–197.
Murotsu T, Matsubara K, Sugisaki H & Takanami M (1981)
Nine unique repeating sequences in a region essential for
replication and incompatibility of the mini-F plasmid. Gene
15: 257–271.
Mushegian AR & Koonin EV (1996) Gene order is not
conserved in bacterial evolution. Trends Genet 12: 289–290.
Myllykallio H, Lopez P, Lopez-Garcia P et al. (2000) Bacterial
mode of replication with eukaryotic-like machinery in a
hyperthermophilic archaeon. Science 288: 2212–2215.
Neuwald AF, Aravind L, Spouge JL & Koonin EV (1999)
AAA+: a class of chaperone-like ATPases associated with the
assembly, operation, and disassembly of protein complexes.
Genome Res 9: 27–43.
Nieto C, Giraldo R, Fernandez-Tresguerres E & Diaz R (1992)
Genetic and functional analysis of the basic replicon of
pPS10, a plasmid specific for Pseudomonas isolated from
Pseudomonas syringae patovar savastanoi. J Mol Biol 223:
415–426.
Nievera C, Torgue JJ, Grimwade JE & Leonard AC (2006)
SeqA blocking of DnaA-oriC interactions ensures staged
assembly of the E. coli pre-RC. Mol Cell 24: 581–592.
ª 2011 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
432
Norais C, Hawkins M, Hartman AL, Eisen JA, Myllykallio H &
Allers T (2007) Genetic and physical mapping of DNA
replication origins in Haloferax volcanii. PLoS Genet 3: e77.
Ogura T & Wilkinson AJ (2001) AAA+ superfamily ATPases:
common structure–diverse function. Genes Cells 6: 575–597.
Ogura T, Niki H, Kano Y, Imamoto F & Hiraga S (1990)
Maintenance of plasmids in HU and IHF mutants of
Escherichia coli. Mol Gen Genet 220: 197–203.
Ohkubo S & Yamaguchi K (1995) Two enhancer elements for
DNA replication of pSC101, par and a palindromic binding
sequence of the Rep protein. J Bacteriol 177: 558–565.
Oka A, Sugimoto K, Takanami M & Hirota Y (1980)
Replication origin of the Escherichia coli K-12 chromosome:
the size and structure of the minimum DNA segment
carrying the information for autonomous replication. Mol
Gen Genet 178: 9–20.
Ortega-Jimenez S, Giraldo-Suarez R, Fernandez-Tresguerres
ME, Berzal-Herranz A & Diaz-Orejas R (1992) DnaA
dependent replication of plasmid R1 occurs in the presence
of point mutations that disrupt the dnaA box of oriR.
Nucleic Acids Res 20: 2547–2551.
Ozaki S, Fujimitsu K, Kurumizaka H & Katayama T (2006)
The DnaA homolog of the hyperthermophilic eubacterium
Thermotoga maritima forms an open complex with a
minimal 149-bp origin region in an ATP-dependent
manner. Genes Cells 11: 425–438.
Ozaki S, Kawakami H, Nakamura K et al. (2008) A common
mechanism for the ATP-DnaA-dependent formation of
open complexes at the replication origin. J Biol Chem 283:
8351–8362.
Pacek M, Konopa G & Konieczny I (2001) DnaA box
sequences as the site for helicase delivery during plasmid
RK2 replication initiation in Escherichia coli. J Biol Chem
276: 23639–23644.
Papp PP, Chattoraj DK & Schneider TD (1993) Information
analysis of sequences that bind the replication initiator
RepA. J Mol Biol 233: 219–230.
Papp PP, Mukhopadhyay G & Chattoraj DK (1994) Negative
control of plasmid DNA replication by iterons. Correlation
with initiator binding affinity. J Biol Chem 269: 23563–
23568.
Park K & Chattoraj DK (2001) DnaA boxes in the P1 plasmid
origin: the effect of their position on the directionality of
replication and plasmid copy number. J Mol Biol 310: 69–81.
Park K, Mukkopadhyay S & Chattoraj DK (1998)
Requirements for and regulation of origin opening of
plasmid P1. J Biol Chem 273: 24906–24911.
Park K, Han E, Paulsson J & Chattoraj DK (2001) Origin
pairing (‘handcuffing’) as a mode of negative control of P1
plasmid copy number. EMBO J 20: 7323–7332.
Pearson CE, Zorbas H, Price GB & Zannis-Hadjopoulos M
(1996) Inverted repeats, stem-loops, and cruciforms:
significance for initiation of DNA replication. J Cell Biochem
63: 1–22.
Pelizon C, Diviacco S, Falaschi A & Giacca M (1996) Highresolution mapping of the origin of DNA replication in the
ª 2011 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
M. Rajewska et al.
hamster dihydrofolate reductase gene domain by
competitive PCR. Mol Cell Biol 16: 5358–5364.
Perri S & Helinski DR (1993) DNA sequence requirements for
interaction of the RK2 replication initiation protein with
plasmid origin repeats. J Biol Chem 268: 3662–3669.
Phadtare S & Inouye M (1999) Sequence-selective interactions
with RNA by CspB, CspC and CspE, members of the CspA
family of Escherichia coli. Mol Microbiol 33: 1004–1014.
Picardeau M, Lobry JR & Hinnebusch BJ (1999) Physical
mapping of an origin of bidirectional replication at the
centre of the Borrelia burgdorferi linear chromosome. Mol
Microbiol 32: 437–445.
Pierechod M, Nowak A, Saari A, Purta E, Bujnicki JM &
Konieczny I (2009) Conformation of a plasmid replication
initiator protein affects its proteolysis by ClpXP system.
Protein Sci 18: 637–649.
Polaczek P, Kwan K & Campbell JL (1998) Unwinding of the
Escherichia coli origin of replication (oriC) can occur in the
absence of initiation proteins but is stabilized by DnaA and
histone-like proteins IHF or HU. Plasmid 39: 77–83.
Rajewska M, Kowalczyk L, Konopa G & Konieczny I (2008)
Specific mutations within the AT-rich region of a plasmid
replication origin affect either origin opening or helicase
loading. P Natl Acad Sci USA 105: 11134–11139.
Rampakakis E, Gkogkas C, Di Paola D & Zannis-Hadjopoulos
M (2010) Replication initiation and DNA topology: the
twisted life of the origin. J Cell Biochem 110: 35–43.
Riber L, Olsson JA, Jensen RB, Skovgaard O, Dasgupta S,
Marinus MG & Lobner-Olesen A (2006) Hda-mediated
inactivation of the DnaA protein and dnaA gene
autoregulation act in concert to ensure homeostatic
maintenance of the Escherichia coli chromosome. Genes Dev
20: 2121–2134.
Richter S, Hess WR, Krause M & Messer W (1998) Unique
organization of the dnaA region from Prochlorococcus
marinus CCMP1375, a marine cyanobacterium. Mol Gen
Genet 257: 534–541.
Robinson NP, Dionne I, Lundgren M, Marsh VL, Bernander R
& Bell SD (2004) Identification of two origins of replication
in the single chromosome of the archaeon Sulfolobus
solfataricus. Cell 116: 25–38.
Rocha EP, Danchin A & Viari A (1999) Universal replication
biases in bacteria. Mol Microbiol 32: 11–16.
Romero J & Lee H (2008) One-way PCR-based mapping of a
replication initiation point (RIP). Nat Protoc 3: 1729–1735.
Roten CA, Gamba P, Barblan JL & Karamata D (2002)
Comparative Genometrics (CG): a database dedicated to
biometric comparisons of whole genomes. Nucleic Acids Res
30: 142–144.
Rowen L & Kornberg A (1978) Primase, the dnaG protein of
Escherichia coli. An enzyme which starts DNA chains. J Biol
Chem 253: 758–764.
Ryan VT, Grimwade JE, Nievera CJ & Leonard AC (2002) IHF
and HU stimulate assembly of pre-replication complexes at
Escherichia coli oriC by two different mechanisms. Mol
Microbiol 46: 113–124.
FEMS Microbiol Rev 36 (2012) 408–434
AT-rich region and the repeated sequences in DNA replication
Ryan VT, Grimwade JE, Camara JE, Crooke E & Leonard AC
(2004) Escherichia coli prereplication complex assembly is
regulated by dynamic interplay among Fis, IHF and DnaA.
Mol Microbiol 51: 1347–1359.
Saha A, Haralalka S & Bhadra RK (2004) A naturally
occurring point mutation in the 13-mer R repeat affects the
oriC function of the large chromosome of Vibrio cholerae
O1 classical biotype. Arch Microbiol 182: 421–427.
Salzberg SL, Salzberg AJ, Kerlavage AR & Tomb JF (1998)
Skewed oligomers and origins of replication. Gene 217: 57–67.
Schaefer C & Messer W (1991) DnaA protein/DNA
interaction. Modulation of the recognition sequence. Mol
Gen Genet 226: 34–40.
Schaper S & Messer W (1995) Interaction of the initiator
protein DnaA of Escherichia coli with its DNA target. J Biol
Chem 270: 17622–17626.
Schnos M, Zahn K, Inman RB & Blattner FR (1988) Initiation
protein induced helix destabilization at the lambda origin: a
prepriming step in DNA replication. Cell 52: 385–395.
Schvartzman JB, Martinez-Robles ML, Hernandez P & Krimer
DB (2010) Plasmid DNA replication and topology as
visualized by two-dimensional agarose gel electrophoresis.
Plasmid 63: 1–10.
Seitz H, Weigel C & Messer W (2000) The interaction
domains of the DnaA and DnaB replication proteins of
Escherichia coli. Mol Microbiol 37: 1270–1279.
Sekimizu K, Bramhill D & Kornberg A (1988) Sequential early
stages in the in vitro initiation of replication at the origin of
the Escherichia coli chromosome. J Biol Chem 263: 7124–7130.
Shaheen SM, Ouimet MC & Marczynski GT (2009)
Comparative analysis of Caulobacter chromosome
replication origins. Microbiology 155: 1215–1225.
Sharma S, Sathyanarayana BK, Bird JG, Hoskins JR, Lee B &
Wickner S (2004) Plasmid P1 RepA is homologous to the
F plasmid RepE class of initiators. J Biol Chem 279: 6027–
6034.
Shon M, Germino J & Bastia D (1982) The nucleotide
sequence of the replication origin beta of the plasmid R6K.
J Biol Chem 257: 13823–13827.
Sibley CD, MacLellan SR & Finan T (2006) The Sinorhizobium
meliloti chromosomal origin of replication. Microbiology
152: 443–455.
Skarstad K, Thony B, Hwang DS & Kornberg A (1993) A
novel binding protein of the origin of the Escherichia coli
chromosome. J Biol Chem 268: 5365–5370.
Slater S, Wold S, Lu M, Boye E, Skarstad K & Kleckner N
(1995) E. coli SeqA protein binds oriC in two different
methyl-modulated reactions appropriate to its roles in DNA
replication initiation and origin sequestration. Cell 82: 927–
936.
Speck C & Messer W (2001) Mechanism of origin unwinding:
sequential binding of DnaA to double- and single-stranded
DNA. EMBO J 20: 1469–1476.
Speck C, Weigel C & Messer W (1999) ATP- and ADP-dnaA
protein, a molecular switch in gene regulation. EMBO J 18:
6169–6176.
FEMS Microbiol Rev 36 (2012) 408–434
433
Stalker DM, Kolter R & Helinski DR (1979) Nucleotide
sequence of the region of an origin of replication of the
antibiotic resistance plasmid R6K. P Natl Acad Sci USA 76:
1150–1154.
Stalker DM, Thomas CM & Helinski DR (1981) Nucleotide
sequence of the region of the origin of replication of the
broad host range plasmid RK2. Mol Gen Genet 181: 8–12.
Stalker DM, Kolter R & Helinski DR (1982) Plasmid R6K
DNA replication. I. Complete nucleotide sequence of an
autonomously replicating segment. J Mol Biol 161: 33–43.
Stenzel TT, MacAllister T & Bastia D (1991) Cooperativity
at a distance promoted by the combined action of two
replication initiator proteins and a DNA bending protein
at the replication origin of pSC101. Genes Dev 5: 1453–
1463.
Stinchcomb DT, Struhl K & Davis RW (1979) Isolation and
characterisation of a yeast chromosomal replicator. Nature
282: 39–43.
Sugimoto K, Oka A, Sugisaki H, Takanami M, Nishimura A,
Yasuda Y & Hirota Y (1979) Nucleotide sequence of
Escherichia coli K-12 replication origin. P Natl Acad Sci USA
76: 575–579.
Sugiura S, Ohkubo S & Yamaguchi K (1993) Minimal essential
origin of plasmid pSC101 replication: requirement of a
region downstream of iterons. J Bacteriol 175: 5993–6001.
Suhan M, Chen SY, Thompson HA, Hoover TA, Hill A &
Williams JC (1994) Cloning and characterization of an
autonomous replication sequence from Coxiella burnetii.
J Bacteriol 176: 5233–5243.
Sutton MD, Carr KM, Vicente M & Kaguni JM (1998)
Escherichia coli DnaA protein. The N-terminal domain and
loading of DnaB helicase at the E. coli chromosomal origin.
J Biol Chem 273: 34255–34262.
Taghbalout A, Landoulsi A, Kern R et al. (2000) Competition
between the replication initiator DnaA and the sequestration
factor SeqA for binding to the hemimethylated
chromosomal origin of E. coli in vitro. Genes Cells 5: 873–
884.
Thony B, Hwang DS, Fradkin L & Kornberg A (1991) iciA, an
Escherichia coli gene encoding a specific inhibitor of
chromosomal initiation of replication in vitro. P Natl Acad
Sci USA 88: 4066–4070.
Tillier ER & Collins RA (2000) The contributions of
replication orientation, gene direction, and signal sequences
to base-composition asymmetries in bacterial genomes.
J Mol Evol 50: 249–257.
Tomizawa J, Sakakibara Y & Kakefuda T (1974) Replication of
colicin E1 plasmid DNA in cell extracts. Origin and
direction of replication. P Natl Acad Sci USA 71: 2260–
2264.
Tomizawa JI, Ohmori H & Bird RE (1977) Origin of
replication of colicin E1 plasmid DNA. P Natl Acad Sci USA
74: 1865–1869.
Torheim NK & Skarstad K (1999) Escherichia coli SeqA protein
affects DNA topology and inhibits open complex formation
at oriC. EMBO J 18: 4882–4888.
ª 2011 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
434
Wei T & Bernander R (1996) Interaction of the IciA protein
with AT-rich regions in plasmid replication origins. Nucleic
Acids Res 24: 1865–1872.
Weigel C & Seitz H (2002) Strand-specific loading of DnaB
helicase by DnaA to a substrate mimicking unwound oriC.
Mol Microbiol 46: 1149–1156.
Weigel C, Schmidt A, Ruckert B, Lurz R & Messer W (1997)
DnaA protein binding to individual DnaA boxes in the
Escherichia coli replication origin, oriC. EMBO J 16: 6574–
6583.
Wickner S (1977) DNA or RNA priming of bacteriophage G4
DNA synthesis by Escherichia coli dnaG protein. P Natl
Acad Sci USA 74: 2815–2819.
Wickner S & Hurwitz J (1975) Interaction of Escherichia coli
dnaB and dnaC(D) gene products in vitro. P Natl Acad Sci
USA 72: 921–925.
Wickner S, Hoskins J, Chattoraj D & McKenney K (1990)
Deletion analysis of the mini-P1 plasmid origin of
replication and the role of Escherichia coli DnaA protein.
J Biol Chem 265: 11622–11627.
Woelker B & Messer W (1993) The structure of the initiation
complex at the replication origin, oriC, of Escherichia coli.
Nucleic Acids Res 21: 5025–5033.
Wu F, Goldberg I & Filutowicz M (1992) Roles of a 106-bp
origin enhancer and Escherichia coli DnaA protein in
replication of plasmid R6K. Nucleic Acids Res 20: 811–817.
Wu F, Wu J, Ehley J & Filutowicz M (1996) Preponderance of
Fis-binding sites in the R6K gamma origin and the curious
effect of the penicillin resistance marker on replication of
this origin in the absence of Fis. J Bacteriol 178: 4965–4974.
ª 2011 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
M. Rajewska et al.
Yamanaka K, Zheng W, Crooke E, Wang YH & Inouye M
(2001) CspD, a novel DNA replication inhibitor induced
during the stationary phase in Escherichia coli. Mol Microbiol
39: 1572–1584.
Yee TW & Smith DW (1990) Pseudomonas chromosomal
replication origins: a bacterial class distinct from
Escherichia coli-type origins. P Natl Acad Sci USA 87:
1278–1282.
Yoshikawa H & Wake RG (1993) 36. Initiation and
termination of chromosome replication. Bacillus subtilis and
Other Gram-Positive Bacteria: Biochemistry, Physiology, and
Molecular Genetics (Sonenshein AL, Hoch JA & Losick R,
eds), pp. 507–528. ASM, Washington, DC.
You Z, Ishimi Y, Mizuno T, Sugasawa K, Hanaoka F & Masai
H (2003) Thymine-rich single-stranded DNA activates
Mcm4/6/7 helicase on Y-fork and bubble-like substrates.
EMBO J 22: 6148–6160.
Zawilak A, Durrant MC, Jakimowicz P, Backert S &
Zakrzewska-Czerwinska J (2003) DNA binding specificity of
the replication initiator protein, DnaA from Helicobacter
pylori. J Mol Biol 334: 933–947.
Zhang CT & Zhang R (2002) Evaluation of gene-finding
algorithms by a content-balancing accuracy index. J Biomol
Struct Dyn 19: 1045–1052.
Zhang R & Zhang CT (2005) Identification of replication
origins in archaeal genomes based on the Z-curve method.
Archaea 1: 335–346.
Zzaman S, Abhyankar MM & Bastia D (2004) Reconstitution
of F factor DNA replication in vitro with purified proteins.
J Biol Chem 279: 17404–17410.
FEMS Microbiol Rev 36 (2012) 408–434