Mol. Cells, Vol. 12, No. 2, pp. 149-157
Molecules
and
Cells
Minireview
KSMCB 2001
Replication of the Lagging Strand: A Concert of at Least 23
Polypeptides
Ulrich Hübscher* and Yeon-Soo Seo1
Institute of Veterinary Biochemistry and Molecular Biology, University of Zürich, Zürich CH-8057, Switzerland;
1
National Creative Research Initiative Center for Cell Cycle Control, Samsung Biomedical Research Institute, Sungkyunkwan
University School of Medicine, Suwon 440-746, Korea.
(Received July 3, 2001; Accepted July 10, 2001)
DNA replication is one of the most important events in
living cells, and it is still a key problem how the DNA
replication machinery works in its details. A
replication fork has to be a very dynamic apparatus
since frequent DNA polymerase switches from the
initiating DNA polymerase α to the processive
elongating DNA polymerase δ occur at the leading
strand (about 8 × 104 fold on both strands in one
replication round) as well as at the lagging strand
(about 2 × 107 fold on both strands in one replication
round) in mammalian cells. Lagging strand replication
involves a very complex set of interacting proteins that
are able to frequently initiate, elongate and process
Okazaki fragments of 180 bp. Moreover, key proteins
of this important process appear to be controlled by Sphase checkpoint proteins. It became furthermore
clear in the last few years that DNA replication cannot
be considered uncoupled from DNA repair, another
very important event for any living organism. The
reconstitution of nucleotide excision repair and base
excision repair in vitro with purified components
clearly showed that the DNA synthesis machinery of
both of these macromolecular events are similar and
do share many components of the lagging strand DNA
synthesis machinery. In this minireview we summarize
our current knowledge of the components involved in
the execution and regulation of DNA replication at the
lagging strand of the replication fork.
Keywords: Dna2; DNA Ligase 1; DNA Polymerase;
DNA Replication; Fen1; Lagging Strand; Okazaki
Fragment Processing; PCNA; RF-C; RNase H1; RPA.
* To whom correspondence should be addressed.
Tel: 41-1-635-54-72/71; Fax: 41-1-635-68-40
E-mail: [email protected]
Introduction
A short replication overview Maintenance of genetic
stability is a key issue for any form of life. Consequently
highly sophisticated mechanisms for maintenance of the
genome were well established before the three kingdoms
of life (bacteria, archaea and eukaryotes) separated. DNA
transactions in eukaryotic cells such as replication, repair
and transcription require a large set of proteins. In all of
these events, complexes of more than 30 polypeptides
appear to function in highly organized and structurally
well defined machines. In the last few years we have
learned that these three essential macromolecular events
have common functional entities and are coordinated by
complex regulatory mechanisms. This can be documented
for replication and repair, for replication and checkpoint
control, for replication and cell cycle control as well as
for replication and transcription (reviewed in Stucki et al.,
2000).
DNA replication is the event leading to the duplication
of DNA in advance of mitosis (or meiosis) and cell
division. It occurs in vivo in an ordered and highly
organized way, in which all enzymes and proteins
involved have their exact roles in a replication complex
called the replisome, which itself is located in so called
nuclear replication factories. Models have been proposed
on how the enzymatic machinery might be spatially
arranged at replication forks. They were based on the idea
that DNA polymerases (pols) dimerize and that the DNA
loops on the lagging strand in a way that the “directionality”
Abbreviations: bp, base pair; DSB, double strand break; MCMs,
minichromosomal maintence proteins; nt, nucleotide; ORC,
origin recognition complex; PCNA, proliferating cell nuclear
antigen; Pol, polymerase; RF-C, replication factor-C; RPA,
replication protein-A.
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Replication of the Lagging Strand in Eukaryotes
for the pols is the same. If one postulates that the replisome is fixed to structures in the nuclear replication
factories, it would thread the DNA through itself. The
assembly and the events in a replisome might occur as
follows (for details see De Pamphiilis, 1996 and articles
therein and Waga and Stillman, 1998).
An initiator protein complex called origin recognition
complex (ORC) is bound to an origin of replication (Fig.
1). ORC has to be activated by other proteins such as
minichromosomal maintenance proteins (MCMs), cell
division cycle proteins (Cdc6 and Cdc7/Dbf4), by
mechanisms such as phosphorylation or possibly other
posttranslational modifications. This leads to the formation of an initiation complex that is able to alter DNA
structures locally in its vicinity presumably by activating
the intrinsic helicase activity of a MCM subcomplex or by
attracting other DNA helicases to the origins. The single
stranded DNA thus produced must be protected and
stabilized by the single stranded DNA binding protein,
called replication protein-A (RPA), which can help to
unwind the DNA by its unwinding activity and, possibly,
through its interactions with DNA helicases and DNA
polymerase α/primase (pol α/primase) which is acting as
the initiating pol. After very limited DNA synthesis, a
DNA polymerase switch from pol α/primase to the processive pol δ holoenzyme occurs, most likely mediated by
the pol auxiliary protein replication factor-C (RF-C). Pol
α/primase itself subsequently acts at the discontinuously
synthesized lagging strand where it initiates Okazaki
fragments. While the pol δ holoenzyme [pol δ, proliferating cell nuclear antigen (PCNA) and RF-C] is
engaged in processive leading strand DNA synthesis, the
situation at the lagging strand is far more complex. Pol δ
holoenzyme is formed for completion of each Okazaki
fragment initiated by pol α/primase. DNA synthesis on
the leading and on the lagging strand are likely coordinated by dimerization of the two processive pol holoenzymes (e.g. two pol δ holoenzymes) possibly upon direct
physical interaction of two pols or via a clamp factor. The
initiator RNA at the lagging strand is removed by the
Dna2 endonuclease and the initiator DNA by Fen1. After
complete DNA synthesis and endonucleolytical processing, the Okazaki fragments are sealed by DNA ligase I.
Topological constraints finally are released by DNA
topoisomerase I. After full replication of an entire replicon unit the two DNA strands can be separated by DNA
topoisomerase II.
Priming by DNA polymerase α/primase and
DNA polymerase switch to DNA polymerase
δ holoenzyme
Pol α/primase is a heterotetramer with molecular masses
of 165, 68−89, 58, and 48 kDa (reviewed in Hübscher et
Fig. 1. DNA polymerases in eukaroytic DNA replication.
Detailed analysis of simian virus 40 (SV40) DNA replication
has led to a model for DNA replication of eukaryotic chromosomal DNA. In the initial step, (1) T antigen (Tag) functions as
a viral origin recognition complex (vORC) comparable to the
cellular ORC found in all eukaryotic cells. (2) Tag recruits the
single stranded DNA binding factor replication protein A (RPA)
and DNA topoisomerase I (topo I) to (3) destabilize and (4)
unwind double stranded origin DNA. (5) Next, Tag loads the
DNA pol α/primase onto the DNA. The primase enzyme of
DNA pol α/primase initiates first leading strand replication.
Thus, these activities of Tag resemble those of several cellular
proteins such as CDC6 (CDC stands for cell division cycle) and
CDC45 that load replication factors onto the chromatin. The
DNA pol α/ primase synthesizes the primer RNA molecules that
are elongated by the intrinsic DNA polymerase activity. (6)
These RNA-DNA molecules then are recognized by RF-C and
PCNA that load DNA pol δ and probably DNA pol ε. The
leading strand is then replicated by DNA pol δ. (7) The Okazaki
fragments on the lagging strand are initiated by DNA pol α/
primase and synthesized by RF-C, PCNA and DNA pol δ and
may be also DNA pol ε. (8) The maturation of the Okazaki
fragments requires a whole set of additional proteins and pol δ
or ε (for a recent reviews see Hübscher et al., 2000).
Reproduced with permission from Elsevier Science.
Ulrich Hübscher & Yeon-Soo Seo
al., 2000). The 48 kDa subunit is the only enzyme that
can start DNA synthesis de novo and contains the catalytic center of the DNA primase. Pol α/primase synthesizes RNA primers of 10 bases while initiating on the
leading strand for continuous synthesis and after every
180 bases on the lagging strand for Okazaki fragments
synthesis (Fig. 2). These RNA primers of 10 bases are
elongated for further 20 DNA bases to about 30 nucleotides by the p165 polymerizing subunit of the heterotetrameric pol α/primase. This initiation event has to happen at the beginning of each Okazaki fragment (about 2 ×
107 times in a mammalian cell per replication event). The
oligonucleotide synthesized by pol α/primase is then utilized by pol δ for processive elongation on both the leading and the lagging strands. Pol δ is more processive since,
it exists as a holoenzyme, composed of three components,
first the heterotetrameric pol δ (reviewed in MacNeill et
al., 2001), second the heteropentameric RF-C (reviewed
in Mossi and Hübscher, 1998) and third the homotrimeric
ring PCNA (revieved in Jonsson and Hübscher, 1997).
The substitution of pol α/primase by the more processive
pol δ holoenzyme is called pol switch. It is absolutely
dependent upon the synthesis of the RNA/DNA primer by
pol α. The pol switch is coordinated and regulated by an
ATP switch catalyzed by the auxiliary protein RF-C
(Maga et al., 2000). It involves a complex network of
interactions among pol α/primase, pol δ, RF-C and RPA
(reviewed in Stucki et al., 2000). Both pol α/primase and
pol δ are perfectly suited for their respective roles: pol
α/primase can initiate synthesis de novo, whereas pol δ,
through its interaction with the processivity factor PCNA,
has the ability to synthesize long stretches of DNA.
Pol α/primase displays a low processivity and, due to
the lack of an intrinsic or associated 3′ → 5′ exonuclease
activity, it is more error-prone than pol δ. It was recently
found that RPA acts as an auxiliary factor for pol α by
playing a dual role. Firstly, it stabilizes the pol α/primer
complex, thus acting as a pol clamp, and secondly, it significantly reduces the misincorporation efficiency by pol
α. Based on these findings it was proposed that RPA is
involved in the regulation of the early events of DNA
synthesis by acting as a "fidelity clamp" for pol α (Maga
et al., 2001).
Elongation and strand displacement by DNA
polymerase δ holoenzyme
As already indicated the inherent infidelity of pol α poses
a problem to the cell, which apparently takes up the risk
of accumulating dangerous mutations during DNA replication. Genetic and biochemical data indicated that the
first 30 nt of each Okazaki fragment synthesized by pol α
that potentially contains mistakes is removed by combined action of Dna2 and Fen1 endonucleases. The re-
151
Fig. 2. Initiation of lagging strand DNA synthesis and DNA
polymerase switch. Initiation of an Okazaki fragment and subsequent elongation includes the following steps: (1) The primase
heterodimer of the four subunits pol α/primase synthesizes an
RNA oligonucleotide of 10 bases, (2) the RNA oligonucleotide
is then elongated by the polymerase subunit of the four subunits
pol α/primase, (3) RF-C binds to the 5′-end of the RNA and
diplaces pol α/primase at the critical length of 30 nucleotides
(10 RNA and 20 DNA), (4) RF-C loads PCNA, (5) PCNA docks
on pol δ, (6) pol δ holoenzyme processively synthesizes DNA
and RF-C leaves the DNA. Processing of the Okazaki fragment
is outlined in Fig. 3.
sulting gap is filled in by the error-free pol δ together
with the accessory proteins RPA, PCNA and RF-C and
sealed by DNA ligase I. The limited strand displacement
activity of pol δ might be important in this process
(Podust et al., 1993). It was recently found that the strand
displacement activity of pol δ can be modulated by the
concerted action of RPA, PCNA and Fen1 (Maga, Villani,
Tillemen, Stucki, Locatelli, Frouin, Spadari, and Hübscher; submitted for publication). The DNA synthesis
across a double stranded DNA region imposes a severe
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Replication of the Lagging Strand in Eukaryotes
block to pol δ, resulting in a reduced incorporation rate
and was highly distributive, depending on multiple dissociation and reassociation events of pol δ. PCNA and RF-C
increase the processivity of the strand displacement activity of pol δ, whereas RPA is able to limit the size of the
displaced fragment to about 30 nt. This block occurs at a
concentration of RPA which was nearly equimolar to the
DNA template, but only if RPA was present before the
displaced strand exceeded the critical size of 30 nt. Fen1
is able to cleave the displaced 30 nt fragment in the presence of RPA, PCNA and RF-C, rendering a fully cut
product. This reaction absolutely requires the catalytic
activity of Fen1, as an active site D86A mutant was unable to process the flap. In sum, it appears that RPA plays
a critical role in regulating the extent of unwinding the
Okazaki fragment (see next paragraph). The block imposed to pol δ by RPA might be important to prevent uncontrolled displacement synthesis catalyzed by pol δ,
which could lead to unnecessary extensive degradation of
a preexisting Okazaki fragment and the formation of excessively long single stranded DNA segments, potentially
forming secondary structures that would be resistant to
cleavage by Fen1. From these results a model for Okazaki
fragment processing emerged: when the growing chain of
an Okazaki fragment elongated by pol δ/PCNA meets the
5′-end of the previously synthesized fragment, pol δ can
invade the double stranded region displacing the RNA/
DNA fragment synthesized by pol α/primase and replacing it with a faithful DNA copy. PCNA can limit the tendency of pol δ to dissociate upon encountering a doublestranded region, thus increasing its processivity. RPA, on
the other hand, would act by ensuring that the displaced
fragment does not exceed a critical size of about 30 nt,
corresponding to the stretch synthesized by pol α/primase. The size of the displaced fragment corresponded to
the length of DNA necessary to bind RPA, suggesting that,
as soon as the single stranded tail generated during strand
displacement by pol δ reaches the right size, it can be
bound by RPA. Such a model also predicts that one flap is
generated per molecule of template and that this flap, due
to its limited size, is able to bind only one molecule of
RPA. The flap structure, which is now stabilized, is then
an ideal substrate for Fen1, which can cleave it, leaving a
nick to be sealed by DNA ligase I. Finally, it is intriguing
to note that many of the proteins involved in Okazaki
fragment DNA synthesis have evolved a similar DNA
substrate specificity: i) the 30 nucleotide size of the
RNA/DNA primer synthesized by pol α/primase has been
shown to be critical for the pol switching process, being
the limiting factor for RF-C binding and subsequent displacement of pol δ, since RF-C has been shown to preferentially bind to a 30 nt long newly synthesized primer
(Maga et al., 2000), ii) RPA preferentially binds to single
stranded DNA tracts of 10 to 30 nt (Blackwell and
Borowiec, 1994), and iii) recent data showed that maxi-
mal endonucleolytic efficiency of Fen1 occurs in the
presence of single stranded DNA flaps from 20 to 40 nt in
length (Hendrickson et al., 2000) suggesting that these
common DNA binding preferences could be essential for
Okazaki fragment processing.
Dna2 and RPA: two new players in processing Okazaki fragments
Extensive work on the maturation of lagging strands suggests that initiator RNA primers of Okazaki fragments are
removed by the combined action of two nucleases, called
RNase HI and Fen1 (reviewed in Bambara et al., 1997;
Lieber, 1997; Waga and Stillman, 1998). Despite the wellestablished in vitro roles of these two enzymes (Waga and
Stillman, 1994), genetic analyses in yeast suggested that
null mutants of either RNase HI or Fen1, as well as
RNase HI/Fen1 double mutants are not lethal (Frank et al.,
1998; Reagan et al., 1995; Sommers et al., 1995), indicating that an additional enzyme for RNA removal is required. The temperature-sensitive growth phenotypes of
Fen1-deficient cells suggested that this additional activity
may still depend in part on a functional Fen1. This enzyme was found in Saccharomyces cerevisiae as Dna2. It
is an essential gene product that was implicated based
upon a number of genetic and biochemical analyses in
chromosomal DNA replication (Braguglia et al., 1998;
Budd and Campbell, 1995; 1997; Fiorentino and Crabtree,
1997; Formosa and Nittis, 1999; Parenteau and Wellinger,
1999). An important clue to the role of Dna2 in Okazaki
fragment processing was its specific association with
Rad27, a yeast homolog of mammalian Fen1 (Budd and
Campbell, 1997). In addition, the temperature-dependent
lethality of Schizosaccharomyces pombe dna2ts mutants
was suppressed by overexpression of genes encoding
subunits of pol δ (cdc1+ and cdc27+), DNA ligase I
(cdc17+), and Fen1 (rad2+) (Kang et al., 2000). These
gene products play a role in elongation or maturation of
Okazaki fragments. Consistent with its implied role in
Okazaki fragment processing, Dna2 possesses DNA unwinding (Bae et al., 1998; Budd and Campbell, 1995) as
well as a single stranded endonuclease activity (Bae et al.,
1998; Budd et al., 2000; Lee et al., 2000). The enzymatic
activity of Dna2 is well suited to remove RNA-initiated
single stranded DNA in Okazaki fragments (Bae and Seo,
2000). Based on these results, it was postulated that the
complete removal of the RNA primer in Okazaki fragments is likely to require the combined action of the two
endonucleases, Dna2 and Fen1 (Bae and Seo, 2000). Recently, we showed that RPA, which may limit the displacement DNA synthesis by pol δ during Okazaki fragment extension, mediates the sequential action of Dna2
and Fen1 during Okazaki fragment processing. RPA was
isolated as a genetic suppressor of the DNA2 mutant allele
Ulrich Hübscher & Yeon-Soo Seo
dna2r405N that encodes a protein lacking the Nterminal 405 amino acids of Dna2 (Bae et al., 2001a;
2001b). The Dna2r405N protein is fully active in vitro,
but cells carrying this mutation show temperature-dependent growth when present in a single copy (Bae et al.,
2001a). Subunits of RPA in multicopy corrected the
growth defect of the mutant strain. Consistent with the
genetic interaction, RPA greatly stimulated endonuclease
activity of Dna2 via stimulating complex formation between Dna2 and substrate DNA. The ternary complex is
an intermediate structure that is eventually converted to
products, since the addition of Mg2+ caused rapid disintegration of the complex. This disintegration is dependent
on functional Dna2 ternary complex formation with
Dna2D657A, since a mutant enzyme devoid of endonuclease activity (Lee et al., 2000) did not disassemble in
the presence of Mg2+. The flap DNA remaining after
Dna2-catalyzed cleavage in the presence of RPA varied in
length from 5 to 7 nt, a size insufficient for the stable association of RPA with single stranded DNA (Iftode and
Borowiec, 1997).
In contrast, the cleavage activity by Fen1 is inhibited by
RPA at an amount sufficient to bind to the substrate suggesting that dissociation of RPA from the 5′ single
stranded DNA tail is an essential step before Fen1 can act
upon the remaining flap structure. Time-course analyses
suggested that the action of Dna2 generates the substrate
used by Fen1 and governs the initial rate of Fen1 activity,
indicating that RPA dictates the sequential action of Dna2
and Fen1. Finally, the products generated by the action of
Dna2 and Fen1 under the dictate of RPA are efficiently
ligated by DNA ligase I. These results led to a novel
mechanism of eukaryotic Okazaki fragment processing
(Bae et al., 2001b). In this model (Fig. 3), (1) the RNA
containing 5′ terminus of an Okazaki fragment is rendered
single-stranded through displacement DNA synthesis
catalyzed by pol δ holoenzyme (Bae and Seo, 2000). (2)
RPA rapidly forms an initial complex with the nascent
flap structure and (3) then recruits Dna2 to form a ternary
complex. This leads to the initial cleavage of RNAcontaining segments by Dna2. Binding of RPA to the displaced end of RNA-containing Okazaki segments does
not allow Fen1 to the flap. In the absence of a functional
Dna2 endonuclease the processing of Okazaki fragment is
blocked and cells are not viable. (4) The cleavage of the
displaced RNA-containing Okazaki fragments by Dna2
results in the disassembly of the ternary complex, allowing both Dna2 and RPA to recycle. The remaining short
flap DNA product can be further processed either by Fen1,
which is loaded onto the DNA through protein-protein
interactions with PCNA (Fen1-dependent) (Gomes and
Burgers, 2000) or alternatively by another nuclease e.g.
ExoI (Qiu et al., 1999), or Dna2 itself (Fen1independent). Recently, analyses of defects associated
with exonuclease-deficient pol δ in the presence of rad27
153
Fig. 3. A model of Okazaki fragment processing in eukaryotes
in which the coordinated action of RPA, Dna2, Fen 1 and DNA
ligase I is required for processing of Okazaki fragments
(adopted from Bae et al., 2001b). The wavy line followed by a
linear line denotes the primer RNA and DNA, respectively. The
direction of Okazaki fragment extension by pol δ is indicated by
an arrow. RF-C is not shown for simplicity. (1) Pol δ holoenzyme extends the newly synthesized Okazaki fragment and displaces the 5′-terminus of the preceding Okazaki fragment. (2)
RPA recognizes and binds to the displaced single stranded flap
containing primer RNA. (3) Dna2 is recruited to form a ternary
complex and removes the RNA-containing segment. (4) Nicked
duplex is generated by the action of either Fen1 or other nucleases such as ExoI after nuclease switching occurs. (5) Finally,
DNA ligase I seals the nicked duplex.
mutant alleles described below suggest that the 3′ → 5′
exonuclease activity of DNA pol δ can substitute for
Fen1 in processing Okazaki fragments. It does this by
creating ligatable nicks, after RNA primers have already
been removed by one of the 5′ nucleases such as RNase H,
ExoI, or Dna2. A ligatable nick could be created as a result of 3′ degradation by the 3′ → 5′ exonuclease activity
of pol δ and realignment of a 5′ end into the gap (Jin et al.,
2001). (5) Finally, the resulting nick is sealed by DNA
ligase I.
The fact that RPA plays a role in Okazaki fragment
processing in vivo is supported by two-independent observations. First, a mutation in DNA2 was identified during a synthetic lethal screen with rfa1Y29H, a tempera-
154
Replication of the Lagging Strand in Eukaryotes
ture-sensitive mutant allele of RFA1 (Steven Brill, personal communication), indicating a functional and/or
physical interaction between Dna2 and RPA. Second, the
32 kDa middle subunit of RPA was crosslinked to RNADNA primers in the lagging strand of replicating SV40
chromosome (Mass et al., 1998). This crosslinking was
observed only with early RNA-DNA primer intermediates
produced by pol α/primase and was not detected with
mature lagging strand products, in good agreement with
the finding that in vitro RPA first binds to the 5′ end of
RNA-DNA primers displaced by pol δ and subsequently
dissociates from the flap. The proposed model in Fig. 3
has also a number of features that could account for several other complicated genetic observations made on
Okazaki fragment maturation: (i) The individual inactivation of either Fen1, ExoI, or Mre11 did not lead to cell
death. However, the inactivation of any two of these enzymes resulted in cell death (Budd et al., 2000), most
likely due to the inefficient generation of nicked duplex
DNA. The overexpression of ExoI in the absence of Fen1
restored the growth of mutant cells at the nonpermissive
temperature (Tishkoff et al., 1997); (ii) A recent in vivo
study demonstrated that double strand break (DSB) repair
in yeast requires both leading and lagging strand DNA
synthesis (Holmes and Haber, 1999). In this study, DSBinduced gene conversion at the MAT locus of S. cerevisiae
was analyzed in mutant strains thermo-sensitive for essential replication factors. Gene conversion, which reflects
successful replication in the MAT locus, decreased 50% in
RAD27 null strains deficient for Fen1, compared to wild
type strains. This suggests that at least 50% of Okazaki
fragment processing occurs in the presence of Fen1 and
that the remaining 50% are carried out through a Fen1independent pathway as shown in our model; (iii) Recent
genetic analyses of rad27 mutant alleles showed that
rad27-n (a nuclease-deficient allele of RAD27) inhibited
the growth of mutant cells, whereas rad27-p (a PCNA
binding defective allele) did not (Gary et al., 1999). Interestingly, intragenic combination of both mutations
(rad27-n,p) reversed the deleterious effect of rad27-n on
cell growth. These results suggest that the interaction of
Rad27-n with PCNA freezes the mutant protein at its
normal functioning position within a multiprotein complex. This may prevent from binding of an alternative
enzyme capable of processing Okazaki fragments to the
incompletely processed DNA. In contrast, Rad27-n,p,
which can not be assembled into the complex, allows
other processing enzymes to bind to the incompletely
processed DNA. Thus other activities besides Fen1 are
required to remove the remaining flap DNA generated by
Dna2 as shown in step 4 in Fig. 3.
Why have eukaryotes evolved this complicated mechanism for the removal of RNA primer? Possibly this
mechanism would permit eukaryotic cells to remove the
DNA beyond the RNA-DNA junction. These small RNA-
DNA primers of about 30 nt are synthesized by the DNA
pol α/primase complex, which lacks a proofreading activity. The removal of the entire RNA-DNA primer region
synthesized by the pol α/primase complex may abrogate
the need to correct any errors inserted by pol δ. Although
the new model above accounts for the essential role of
Dna2, this model is compatible with the possibility that
the two nuclease Dna2 and Fen1 with RNase HI constitute parallel pathways under some circumstances. If
displacement DNA synthesis by pol δ is inefficient, for
ex-ample, Okazaki fragments could be susceptible to the
action of RNase HI and Fen1 before the upstream polymerase arrives. In this case, the 5′ → 3′ exonuclease activity of Fen1 could contribute to the removal of the RNA
initiated primer with the aid of RNase HI (Waga and
Stillman, 1994). If displacement DNA synthesis by pol δ
generates a short flap insufficient for RPA binding, then
the removal of the RNA-containing flap may depend
solely on Fen1 alone.
Control of lagging strand DNA replication
Due to the complex logistics of the lagging strand DNA
synthesis it is crucial that control mechanisms guarantee a
faithfull copy of the lagging strand during DNA
replication. Members of the lagging strand machinery are
targets for checkpoint control and for important posttranslational modifications such as phosphorylation and
acetylation.
Pol α/primase, RF-C, pol δ and RPA are phosphorylated by various kinases (reviewed in De Pamphilis 1996
and various articles therein). The DNA primase has been
found to have a role in coupling DNA replication to DNA
damage response (Marini et al., 1997) and the activation
of the replication checkpoint through RNA synthesis by
primase is another hint that this enzyme is a target for
control (Michael et al., 2000). Furthermore the pol α/
primase complex can couple DNA replication to cellcycle progression and to DNA-damage response (Foiani
et al., 1997).
Members of the Rad protein family are involved in the
DNA damage checkpoint and/or in DNA replication
checkpoint control. These proteins mediate a signal to the
cell cycle checkpoint machinery upon stalling of the
replication fork or upon DNA damage (Lowndes and
Murguia, 2000). This leads to a delay of the cell cycle,
until the stalling of the replication fork is released, or
until DNA damage is repaired, thereby preserving genomic integrity. It was found that Rad17 homologs from
human and yeasts (S. cerevisiae and S. pombe) interact
genetically and physically with small subunits of the
clamp loader complex RF-C. This interaction is supposed
to play a role in DNA damage and DNA replication
checkpoint control (Green et al., 1999; Naiki et al., 2000;
Ulrich Hübscher & Yeon-Soo Seo
155
Table 1. Proteins and their duties at the lagging strand of the eukaryotic replication fork.
Protein (polypeptides)
Function
Pol α/primase (4)
Synthesis of RNA-DNA primers
Pol δ (4)
(a) Elongation of RNA-DNA primers, (b) Generation of flap DNA by displacement DNA synthesis
RF-C (5)
(a) Polymerase switching, (b) Loading / unloading of PCNA
PCNA (3)
Clamp to increase processivity of pol δ
Fen1 (1)
Generation of ligatable nicks by cleaving at the flap junction
Dna2 (1)
Cleavage of flap DNA containing primer RNA
RNase H1 (1)
Removal of primer RNA
(a) Specific priming by pol α/primase, (b) Fidelity clamp for pol α, (c) Extension of newly synthesized
Okazaki fragments by pol δ, (d) Dna2-catalyzed cleavage of flap DNAs containing RNA primers,
(e) Regulation of displacement DNA synthesis by pol δ.
Sealing of nicks
RPA (3)
DNA ligase I (1)
Shimamura et al., 1998). Since the Rad17 has homology
to the conserved region of RF-C it is conceivable that
under genotoxic stress during DNA replication the checkpoint protein Rad17 can replace certain subunits in the
heteropentameric RF-C stopping or at least slowing down
DNA replication, thus allowing DNA repair to occur
(Dahm, Freire, Maga and Hübscher, manuscript in
preparation).
We have recently found that the transcriptional coactivator p300 forms a complex with Fen1 and acetylates
Fen1 in vitro (Hasan et al., 2001). Fen1 acetylation is also
evident in vivo and is enhanced upon UV treatment of
human cells. Remarkably, acetylation of the Fen1 Cterminus by p300 significantly reduces DNA binding and
nuclease activity of Fen1. This finding was not unexpected since the C-terminal basic domain of Fen 1 is essential
for binding to DNA (Stucki et al., 2001). PCNA is able to
stimulate both acetylated and unacetylated Fen1 activity
to the same extent. These data identified acetylation as a
novel regulatory modification of Fen1 and suggested that
p300 is not only a component of the chromatin remodelling machinery but might also play a critical role in
regulating DNA metabolic events. In relation to the regulation of Okazaki fragment DNA synthesis it is conceivable that down regulation of Fen1 by acetylation after a
genotoxic event is sought to prevent premature Okazaki
fragment processing by Fen1. This would guarantee the
full removal of the initiator DNA synthesized by pol α,
which would then be a significant advantage for cells to
maintain genomic integrity since this mechanism is independent of a mismatch correcting system that removes
any errors inserted in the RNA/DNA primers.
Acknowledgments U. Hübscher has continuously been supported by the Swiss National Science Foundation, the Zürich
Cancer League and the Kanton of Zürich. Y.-S. Seo was
supported by a grant from the Creative Research Initiatives Pro-
gram of the Korean Ministry of Science and Technology. We
thank Ghislaine Henneke for critical reading of the manuscript.
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