Molecular Microbiology (2002) 43(4), 823–834
MicroReview
A novel family of regulated helicases/nucleases from
Gram-positive bacteria: insights into the initiation of
DNA recombination
Frédéric Chédin† and Stephen C. Kowalczykowski*
Sections of Microbiology and of Molecular and
Cellular Biology, University of California, Davis,
CA 95616-8665, USA.
Introduction
Homologous DNA recombination can be defined at a
molecular level as the exchange of genetic information
between two homologous DNA molecules. It is critically
important in fundamental processes such as the repair of
genotoxic lesions, the creation of genetic diversity, the
proper segregation of chromosomes and the restart
of stalled DNA replication forks. Detailed genetic and
biochemical analyses in the bacterium Escherichia coli
and in the yeast Saccharomyces cerevisiae led to the
realization that DNA recombination can be divided into
four successive steps, with each step requiring the coordinated action of a large number of proteins (for a
review, see Kowalczykowski et al., 1994; Pâques and
Haber, 1999).
At the heart of homologous recombination lies the DNA
strand-exchange step. This step involves the alignment of
two homologous DNA molecules and the exchange of
their complementary strands to produce a joint DNA molecule. In E. coli, the main protein involved at this step is
the RecA protein. Structural and functional homologues of
RecA protein have been discovered throughout Eukarya
and Archaea, thus emphasizing their critical role in all
forms of cellular life (Bianco et al., 1998; Seitz et al.,
1998). In all cases, DNA strand-exchange proteins are
able to bind to single-stranded DNA (ssDNA) in a cooperative way, forming a highly ordered nucleoprotein filament in which the DNA is extended. This filament is the
active species of the protein: it carries out the search for
a homologous duplex DNA partner, pairs the two molecules and catalyses the exchange of their strands.
Accepted 31 October, 2001. *For correspondence. E-mail
[email protected]; Tel. (+1) 530 752 5938; Fax (+1)
530 752 5939. †Present address: Norris Comprehensive Cancer
Center, University of Southern California – School of Medicine, Los
Angeles, CA 90033, USA.
© 2002 Blackwell Science Ltd
Despite some biochemical differences between the
various known DNA strand-exchange proteins, ssDNA is
universally used by all of them to initiate the homologous
pairing and DNA strand-exchange events, although other
modes of initiation are possible (Mazin and Kowalczykowski, 1999; Zaitsev and Kowalczykowski, 2000).
Since DNA in cells is normally found in the doublestranded B form (dsDNA), homologous recombination
calls for a first step in which the dsDNA is processed to
produce a suitable substrate – ssDNA – for the binding of
DNA strand-exchange proteins. This first phase is referred
to as the initiation phase and will be the main focus of this
review. Initiation sites include both dsDNA breaks, either
‘normal’ physiological breaks (e.g. programmed during
the course of meiosis or formed at a stalled replication
fork) or pathological breaks (e.g. caused by exposure to
g-radiation or certain chemicals), and single-strand DNA
gaps (e.g. such as those which exist after the collapse
and restart of a DNA replication fork) (Kowalczykowski,
2000). This review will primarily discuss dsDNA breaks as
initiation sites.
Once a dsDNA break has been introduced into DNA,
the creation of ssDNA can be achieved by several means
(Fig. 1). DNA helicases can catalyse the separation of the
duplex DNA into its individual strands. Initiation by helicases has been documented in vivo as well as in vitro
in bacteria (Thaler et al., 1989; Harmon and Kowalczykowski, 1998; Churchill et al., 1999; Cohen and
Sinclair, 2001). Double-stranded exonucleases degrading
one strand of the duplex can also be used to produce a
single-stranded region and such processing has also
been documented both in vivo and in vitro in different
organisms (Little, 1967; Kolodner et al., 1994). Another
possibility is to associate the activities of a DNA helicase
and a single-stranded DNA nuclease to produce the
desired ssDNA region. This can be achieved by the
coupling of the action of two distinct enzymes, each contributing one of the two activities (Lovett et al., 1988), or
from the action of one enzyme containing both helicase
and nuclease activities. The RecBCD enzyme of E. coli
has, for a number of years, been the prototype for this
latter category and has been the focus of considerable
824 F. Chédin and S. C. Kowalczykowski
Fig. 1. Creation of single-stranded DNA from a double-stranded
DNA break (top) can be achieved by the action of either DNA
helicases (e.g. E. coli RecBC or RecQ proteins – left), doublestranded DNA-specific exonucleases (e.g. E. coli RecE protein or
phage lambda Reda protein – middle), or a combination of
helicases and nucleases (e.g. E. coli RecQ–RecJ proteins,
RecBC–RecJ proteins, or RecBCD enzyme and c – right). The
newly created region of ssDNA will then be used to initiate
homology-dependent pairing and DNA strand exchange, a reaction
catalysed by the RecA protein.
attention. Recently, however, a novel class of enzymes
that are structurally different from the RecBCD enzyme,
but that carry both helicase and nuclease activities,
has emerged from studies of DNA recombination in the
Gram-positive bacterium Bacillus subtilis. This discovery
allowed the E. coli paradigm to be made universal among
Bacteria and to highlight interesting differences in the
use of such enzymes to initiate DNA recombination. This
review will briefly summarize our current knowledge of
the RecBCD class of enzymes (for reviews, see also
Kowalczykowski et al., 1994; Myers and Stahl, 1994) and
will then emphasize the recent developments and their
implications for our understanding of DNA recombination.
Of central importance for this review will also be a class
of short DNA sequences, collectively referred to as Chi
sequences, that have the unique ability to regulate the
activities of their own cognate helicase/nuclease enzyme
and thereby effect DNA recombination.
The RecBCD class of enzymes
The RecBCD enzyme is a large (330 kDa) heterotrimeric
complex composed of the RecB, RecC and RecD proteins
that initiates the main pathway of recombination in E. coli.
Mutations in the recB or recC genes cause about a 100fold decrease in the frequency of conjugal recombination
(Howard-Flanders and Theriot, 1966; Emmerson, 1968)
and strongly sensitize cells to DNA-damaging treatments
such as UV light or X-ray radiation (Sargentini and
Smith, 1986). The RecBCD enzyme has long been known
to be a potent ATP-dependent dsDNA exonuclease,
often referred to as ExoV (Telander-Muskavitch and
Linn, 1981). Subsequent studies showed that RecBCD is
also a processive DNA helicase, for which unwinding
is coupled to exonucleolytic degradation of the DNA.
Notably, RecBCD enzyme has the unique ability to load
onto a blunt or near-blunt dsDNA end and to unwind more
than 30 kilobases per binding event (Taylor and Smith,
1985; Roman et al., 1992; Bianco et al., 2001). Its
degradative function, however, seems to be at odds with
the view that RecBCD is involved in initiating DNA recombination, a process which requires preservation of DNA.
This paradox was solved by the discovery that the nuclease properties of the enzyme are regulated upon recognition of a specific DNA sequence by the translocating
RecBCD enzyme. This sequence, called Chi (c; Lam
et al., 1974; Stahl et al., 1975), corresponds to the
octamer 5¢-GCTGGTGG-3¢ (Smith et al., 1981; Bianco
and Kowalczykowski, 1997). Recognition of c by RecBCD
is polar: c will only be recognized by a RecBCD molecule
travelling through dsDNA from right to left as written
above. This recognition event elicits a number of changes
in the behaviour of the RecBCD enzyme (Fig. 2, left).
Upon recognition, the vigorous 3¢ to 5¢ nuclease that
was present before c is strongly downregulated, while a
weaker 5¢ to 3¢ nuclease activity is activated on the
opposite strand (Dixon and Kowalczykowski, 1991, 1993;
Anderson and Kowalczykowski, 1997a). After recognition
of c by the translocating RecBCD enzyme and its
eventual dissociation from the DNA, the modifications
described above enable the RecBCD enzyme to produce
a dsDNA molecule with a 3¢-ssDNA tail terminating at c
that can be bound by the RecA protein to initiate the
homologous pairing phase of genetic recombination.
Recent studies also showed that the c-activated RecBCD
enzyme directly facilitates the loading of the RecA protein
onto the c-terminated ssDNA. This ensures maximal
participation of this c-protected ssDNA in the process of
genetic recombination (Anderson and Kowalczykowski,
1997b) and effectively provides for co-ordination of the
two first steps of this process. The mechanism by which
c recognition is translated into a change of enzymatic
activity has been the subject of much debate and is still
not clearly understood (for reviews, see Kowalczykowski
et al., 1994; Myers and Stahl, 1994). It appears, however,
that the RecD subunit of the complex plays a major
role in this regulation. Indeed, although dissociation of
the RecD subunit does not occur upon c recognition
(Dohoney and Gelles, 2001), the RecBC enzyme displays
most of the recombinationally important biochemical
activities of the c-modified RecBCD enzyme. Most
notably, it simply functions as a DNA helicase lacking the
© 2002 Blackwell Science Ltd, Molecular Microbiology, 43, 823–834
A novel family of regulated helicases/nucleases
825
Fig. 2. Processing of c-containing DNA by
E. coli RecBCD enzyme (left) and B. subtilis
AddAB enzyme (right). See text for details.
potent nuclease activity of the RecBCD enzyme, it is
unable to respond to c, but it retains the ability to load the
RecA protein onto the 3¢-terminated DNA strand (Dixon
et al., 1994; Anderson et al., 1997; Churchill et al., 1999).
Although the RecBCD enzyme and the regulation of its
activities by c have long been considered a model for
understanding the initiation of DNA recombination in Bacteria, evidence supporting this assumption was, until very
recently, quite limited. Because of the advance of genomic
sequencing, we can now ascertain the existence of true
RecBCD homologues (i.e. containing three subunits
showing extensive amino acid homology to RecB, RecC
and RecD) in several genera, encompassing a large part
of the eubacterial tree (Fig. 3). Despite this prevalence,
however, it appeared that a functional interaction between
the E. coli c sequence and other RecBCD enzymes was
preserved only in enteric bacteria that are closely related
to E. coli (McKittrick and Smith, 1989). This suggested
either that the RecBCD–c interaction was restricted to a
small group of microbes or that functionally equivalent
sequences unrelated to the canonical E. coli c sequence
were used in different organisms. Subsequent work
showed that this latter view was correct. Short specific
DNA sequences (5–8 bp) that protect dsDNA from exonucleolytic degradation in the bacteria Lactococcus lactis
(Biswas et al., 1995), Haemophilus influenzae (Sourice
et al., 1998) and Bacillus subtilis (Chédin et al., 1998)
have been identified. In all cases, those putative c
sequences were believed to interact with, and regulate,
an enzyme that possesses both helicase and exonuclease activity. The H. influenzae enzyme is a clear homologue of RecBCD. However, for the two Gram-positive
bacteria L. lactis and B. subtilis, the corresponding
enzyme seems to belong to a novel class of enzymes that
are functionally, but not structurally, homologous to the
RecBCD class of enzymes.
© 2002 Blackwell Science Ltd, Molecular Microbiology, 43, 823–834
The AddAB class of enzymes
It has long been known that a strong ExoV activity is
present in crude extracts of B. subtilis (Chestukhin et al.,
1972; Ohi and Sueoka, 1973). Additionally, several mutant
strains deficient for this ATP-dependent deoxyribonuclease (add locus) activity were identified (Doly et al.,
1974). Initial purification attempts failed to identify unambiguously the proteins responsible for this activity, but
confirmed that this protein shares the classic requirements for an energy cofactor (ATP) and a divalent cation
(Mg2+) with other ExoV enzymes (Doly and Anagnostopoulos, 1976; Shemyakin et al., 1979). The cloning and
sequencing of two genes encoding B. subtilis ExoV, addA
and addB, were reported by Kooistra and colleagues
(Kooistra et al., 1988; Kooistra and Venema, 1991). These
two genes are organized as an operon and, interestingly,
are upregulated during competence development as
part of a competence-specific regulon (Hamoen et al.,
1998) that also includes the recA gene (Haijema et al.,
1996a).
Structure of the complex
In B. subtilis, addA and addB encode the two subunits of
the AddAB enzyme (135 and 141 kDa respectively). The
AddA subunit shows regions of homology to the RecB
subunit of the RecBCD enzyme. These regions of homology are shared by a large family of DNA helicases and
define an ATP-binding motif and five other potential helicase motifs. A recently identified nuclease domain present
on the C-terminus of the E. coli RecB protein (Yu et al.,
1998) is also present at the C-terminus of AddA (Haijema
et al., 1996b), further extending the structural conservation between RecB and AddA. In contrast, the AddB
subunit does not show substantial homology to the other
826 F. Chédin and S. C. Kowalczykowski
Fig. 3. Distribution of the RecBCD and AddAB enzymes in eubacterial genomes. Groups containing RecBCD homologues are indicated in
blue, while groups containing AddAB homologues are indicated in green. The schematic tree was based upon 16S ribosomal RNA sequence
comparisons. Numbers in parentheses indicate the number of species containing a RecBCD or AddAB homologue in each group. The species
containing a homologue are given.
known proteins, including RecC or RecD, except for a
second putative ATP-binding motif and a second copy of
the same nuclease motif carried by AddA (see below).
Direct homologues of the B. subtilis AddAB complex have
now been identified by sequence homology and/or direct
function in at least 12 different bacterial species, including three other bacilli (B. anthracis, B. stearothermophilus
and B. halodurans), Lactococcus lactis, Clostridium
acetobutylicum and Clostridium difficile, Enterococcus
faecalis, Staphylococcus aureus and various streptococci (S. pyogenes, S. mutans and S. pneumoniae)
(Fig. 3). All of these organisms belong to the
Bacillus/Clostridium subdivision of the Gram-positive bacteria, which seems to be a specialized ‘niche’ where the
AddAB class of enzymes thrives. It is interesting to note,
however, that despite conservation in many species, AddB
subunits show a relatively low level of homology (25–
40% identity) to each other when compared with other
conserved proteins such as RecA. Numerous data,
both genetic and biochemical, clearly demonstrate that
at least the AddAB enzymes of B. subtilis and L. lactis
are composed of only two subunits (see below). However, inspection of the recently sequenced B. subtilis
genome reveals the presence of a putative homologue
of the E. coli RecD protein (encoded by the gene
yrrC). This gene, located more than 1 megabase
away from the addAB operon, is also conserved in L.
lactis (S. D. Ehrlich, personal communication). However, the presence of this gene might be unrelated as it
appears that a family of RecD homologues are present
even in organisms that are clearly devoid of RecBC or
AddAB counterparts, such as Deinococcus radiodurans
(encoded by the gene DR1902) or the archaeon
Methanococcus jannaschii (encoded by the gene
MJ1519). To date, it is therefore unclear whether yrrC
encodes a bona fide RecD homologue or whether this
gene product is involved in a different aspect of DNA
metabolism.
© 2002 Blackwell Science Ltd, Molecular Microbiology, 43, 823–834
A novel family of regulated helicases/nucleases
Organism
Sequence
Size (nt)
Enzyme
Reference
E. coli
H. influenzae
B. subtilis
L. lactis
5¢-GCTGGTGG-3¢
5¢-GNTGGTGG-3¢
5¢-AGCGG-3¢
5¢-GCGCGTG-3¢
8
8 (degenerate)
5
7
RecBCD
RecBCD
AddAB
AddAB
Smith et al. (1981)
Sourice et al. (1998)
Chédin et al. (1998)
Biswas et al. (1995)
Involvement of AddAB in homologous
DNA recombination
B. subtilis and L. lactis are the only two organisms for
which there is substantial information regarding the role
of their AddAB enzyme in cellular DNA metabolism [for
convenience, the homologue of the B. subtilis AddAB
enzyme in L. lactis, originally designated RexAB (for
recombination exonuclease) (El Karoui et al., 1998) will
be referred to in a generic sense as AddAB]. In both B.
subtilis and L. lactis, inactivation of addA or addB leads
to increased sensitivity to DNA-damaging treatments,
lower viability and reduced frequencies of homologous
recombination, as measured by phage transduction or
conjugation (Alonso et al., 1993; Kooistra et al., 1997; El
Karoui et al., 1998). These phenotypes closely mimic
those observed for E. coli recBCD– cells. In B. subtilis,
AddAB defines one of the main pathways of homologous
recombination (Alonso et al., 1991, 1993). Interestingly,
expression of B. subtilis or L. lactis AddAB in E. coli allows
nearly full complementation of a recBC– strain for UV sensitivity and conjugal recombination (Kooistra et al., 1993;
El Karoui et al., 1998), establishing that AddAB and
RecBCD enzymes are performing largely overlapping
functions and that no other host-specific factors are
required for their function.
Chi sequences in other Gram-positive organisms
Using a potent dsDNA exonuclease to initiate DNA recombination, a process that is designed to repair and maintain the integrity of the genetic information, requires that
the exonucleolytic activity be regulated in some way. As
described above, the E. coli RecBCD enzyme recognizes
and responds to a specific 8 bp DNA sequence while
translocating through DNA. This interaction transforms
the RecBCD enzyme from a degradative nuclease to a
recombinogenic nuclease by both attenuating and changing the polarity of DNA degradation. Several independent
lines of evidence argue convincingly that B. subtilis and
L. lactis also possess specific DNA sequences that are
actual homologues of the E. coli c sequence (they will be
referred to as cBs and cLl respectively). These sequences,
when present on dsDNA, protect DNA from exonucleolytic
degradation mediated by the corresponding RecBCD or
AddAB enzyme in vivo (Dabert et al., 1992; Biswas et al.,
1995; Chédin et al., 1998). Analysis of their composition
© 2002 Blackwell Science Ltd, Molecular Microbiology, 43, 823–834
827
Table 1. List of all known Chi sequences in
their respective organisms.
and distribution in their respective genomes reveals some
additional similarities to the E. coli sequence: these
sequences are short (5 bp for cBs and 7 bp for cLl; Table 1),
are rich in G residues, are strongly over-represented
and preferentially co-oriented with respect to DNA
replication (Tracy et al., 1997; Chédin et al., 1998; El
Karoui et al., 1999). Direct evidence for an interaction
between AddAB and cLl was obtained in vivo for L.
lactis: the L. lactis AddAB, when expressed in E. coli
recBCD– cells, promotes the degradation of dsDNA
unless the DNA carries a lactococcal cLl site (El Karoui
et al., 1998). It was also shown that cLl actually functions
as an AddAB-dependent recombination hot-spot (El
Karoui et al., 1998), thus completing the analogy between
the original E. coli c site and its counterpart. In B. subtilis,
the interaction between the AddAB enzyme and its
cognate sequence has been studied in detail in vitro
(Chédin et al., 2000).
Interaction of the B. subtilis AddAB enzyme with its
cognate chi sequence
In vitro, the B. subtilis AddAB enzyme shows activities
that are hallmarks of the RecBCD enzyme: it is both
a powerful and processive DNA helicase, a potent
dsDNA exonuclease and an ATPase (Haijema et al.,
1996c; Kooistra et al., 1997; F. Chédin and S. C. Kowalczykowski, unpublished observations). An interesting distinction between the AddAB enzyme and the RecBCD
enzyme, however, is that while degradation by the
RecBCD enzyme shows a strong preference for the 3¢terminated strand at the enzyme’s entry point (Dixon and
Kowalczykowski, 1993), the degradation of DNA by
AddAB is nearly symmetrical on the two strands (Fig. 2,
right). This behaviour is consistent with the fact that the
AddAB enzyme contains two putative nuclease domains,
as shown in Fig. 4, while the RecBCD enzyme contains
only one. Interaction and recognition of cBs by the AddAB
enzyme only occurs in one orientation; the enzyme must
approach the 5 bp sequence 5¢-AGCGG-3¢ from the 3¢
side (as written here) for recognition to occur (Chédin
et al., 2000). This orientation specificity is also found for
L. lactis despite initial reports documenting a lack of orientation specificity (El Karoui et al., 2000) and is in agreement with the behaviour of the E. coli RecBCD enzyme.
In B. subtilis, when recognition occurs (under optimal
828 F. Chédin and S. C. Kowalczykowski
Fig. 4. The AddAB enzyme carries two homologous nuclease domains.
A. Alignment of the nuclease domain found in the E. coli RecB protein with homologous domains found in various AddA proteins. The
asterisks indicate the three residues that were shown to be critical for nuclease activity (Yu et al., 1998; Wang et al., 2000).
B. Alignment of the second putative nuclease domains present at the C-terminus of various AddB proteins. Red indicates full conservation,
blue indicates conservative substitutions. The boxes indicate domains where homologous residues tend to cluster. nd indicates that the exact
co-ordinate of the leftmost residue is not known precisely (sequencing in progress). Alignment and consensus building was carried out using
the CLUSTALW program. Preliminary sequence data were obtained from The Institute for Genomic Research website at http://www.tigr.org.
conditions, 30% of the time), the potent 3¢–5¢ nuclease
activity is completely attenuated, while the 5¢–3¢ nuclease
activity is unaffected. This attenuation occurs precisely
one nucleotide before the cBs sequence, irrespective of
the strength of the nuclease activity before cBs, which
suggests that the recognition event is very specific. After
recognition and attenuation have occurred, unwinding
resumes. The AddAB enzyme then behaves solely as a
5¢ to 3¢ nuclease, thus allowing for the formation of a
3¢-terminated single-stranded DNA overhang, a type of
intermediate that is used universally to initiate the
process of homologous DNA recombination.
AddAB compared with RecBCD:
similarities and differences
The most striking difference between the AddAB and the
RecBCD enzymes is the fact that, despite their similar biochemical properties, the two enzymes have a very different subunit composition. The AddA subunit, based on
structural homology, is a direct homologue of the RecB
subunit, which can, by itself, translocate along ssDNA and
displace short oligonucleotides (Phillips et al., 1997). It is
therefore believed that the RecB subunit, and, hence, the
AddA subunit, is the helicase of the complex, using the
energy derived from ATP hydrolysis to move along DNA
and to catalyse its unwinding (Boehmer and Emmerson,
1992). Correspondingly, mutants of the RecB or the AddA
subunit that cannot bind or hydrolyse ATP are completely
defective for DNA recombination (Hsieh and Julin, 1992;
Haijema et al., 1996d).
Knowing that the AddAB enzyme degrades both
strands of the DNA duplex and that each of its subunits
carries a conserved nuclease domain, it is tempting to
speculate that each strand is being specifically degraded
by one subunit. Because of its homology with RecB,
which cross-links with the 3¢-terminated strand at the
enzyme’s entry point (Ganesan and Smith, 1993; Farah
and Smith, 1997) and which is known to translocate in a
3¢ to 5¢ direction (Phillips et al., 1997; Bianco and Kowalczykowski, 2000), it is likely that the AddA subunit is
responsible for the 3¢ to 5¢ degradation, an activity that
can be switched off by recognition of c. AddB, on the other
hand, is proposed to degrade the opposite strand, with a
© 2002 Blackwell Science Ltd, Molecular Microbiology, 43, 823–834
A novel family of regulated helicases/nucleases
5¢ to 3¢ polarity; this activity, however, is not subject to
regulation by c. From this analogy, it follows that the AddB
subunit probably incorporates functions normally carried
by the RecC and RecD subunits. This probably includes
stimulation of the DNA helicase activity of AddA in a
manner analogous to stimulation of the weak helicase
activity of the RecB subunit by the RecC subunit and,
even further, by the RecD subunit (Phillips et al., 1997).
Finally, the presence of AddB allows the complex to recognize and to respond to its cognate c sequence, which
suggests that the AddB subunit is a functional homologue
of both the RecC subunit (involved in c recognition; Arnold
et al., 1998) and the RecD subunit (involved in the regulatory response to c; Churchill et al., 1999). Precisely how
recognition of c by the AddAB enzyme is translated into
a change of enzymatic activity is presently unknown.
One of the recently discovered functions of the
RecBCD enzyme is to direct the loading of the RecA
protein onto the c-containing, 3¢-terminated ssDNA
(Anderson and Kowalczykowski, 1997b), a function that
is also shared by the RecBC enzyme (Churchill et al.,
1999). The significance of this loading phenomenon pertains to the fact that the RecA protein is at a strong disadvantage compared with the SSB protein for binding to
ssDNA. Thus, systems that reverse this competition in
favour of RecA protein have evolved and play an important role in the initiation of DNA recombination. Such alternative systems actually exist in E. coli, where they involve
the RecF, RecO and RecR proteins. These proteins allow
RecA protein to compete better with the SSB protein for
binding to ssDNA and limit the extension of the nucleoprotein filament to the appropriate regions (Umezu et al.,
1993; Umezu and Kolodner, 1994; Webb et al., 1997). To
date, the co-ordinated loading of RecA by the AddAB
enzyme has not been demonstrated for the B. subtilis
system (F. Chédin and S. C. Kowalczykowski, unpublished observations). However, a heterologous combination of E. coli RecA protein and B. subtilis AddAB enzyme
was used instead of the proper intraspecies combination,
which could have affected the final result. It is indeed
known that optimal levels of recombination require the
presence of RecA protein and RecBCD enzyme from the
same species (Rinken et al., 1991; de Vries and Wackernagel, 1992). If this negative result were to be confirmed
using a homologous combination of proteins, it could
strongly suggest that other systems (such as the RecF, O
and R proteins) provide the essential RecA-loading function in B. subtilis. In this regard, it is interesting to note
that the RecF pathway also exists in B. subtilis, and it
seems to play a much more prominent role in this organism, as judged by analysis of strains impaired for the recF
or recO genes (Alonso et al., 1991, 1993; Fernandez
et al., 1999).
© 2002 Blackwell Science Ltd, Molecular Microbiology, 43, 823–834
829
Conclusion
Regulated helicases/exonucleases such as the RecBCD
or the AddAB enzyme are now seen to be widespread
among Eubacteria, where they are expected to play
an important role in the initiation of DNA recombination
and recombination-dependent DNA replication. One can
wonder, however, why this solution, which involves a
voracious dsDNA nuclease and the much-needed regulatory switch to ‘domesticate’ them (Myers and Stahl,
1994), has been so successful when other, seemingly
easier, routes could have been used to create the appropriate substrates for recombination (Fig. 1).
One answer to this puzzle might stem from an evolutionary advantage conferred to the cells by the presence
of a strong double-stranded DNA exonuclease activity.
Indeed, in E. coli, the recD gene, and hence the activity
of the ExoV of RecBCD, is critically required for survival
under conditions where a large number of unrepairable
double-stranded DNA breaks are being generated (Michel
et al., 1997; Seigneur et al., 1998). Under such conditions,
which could be created by saturation of the recombination machinery upon growth under extremely adverse
conditions, or otherwise in a recA– genetic background,
broken chromosome arms must be degraded by the
exonuclease activity of RecBCD if cells are to survive
(Skarstad and Boye, 1993; Uzest et al., 1995; Kuzminov
and Stahl, 1997). An illustration of this is found in Photobacterium profundum, a marine bacterium whose ability
to grow at elevated pressure is strictly dependent on the
recD gene, indicating that ExoV activity is required under
these extreme growth conditions (Bidle and Bartlett,
1999). Another benefit from the presence of ExoV is the
prevention of DNA recombination between diverged
sequences. Homologous recombination indeed increases
100- to 1000-fold in a recD– strain as a result of the failure
to degrade aberrant, mismatch-containing, recombination
intermediates (Zahrt and Maloy, 1997; Stambuk and
Radman, 1998). Additionally, it is commonly known that
ExoV provides for a defence against intruding linear
dsDNA molecules (Russel et al., 1989), although it is
worth mentioning that in many cases this protection is
far from perfect (see below). It therefore appears that
although the RecBC enzyme is able to catalyse the initiation phase of DNA recombination, the additional presence of the RecD subunit might enable the cells to cope
with the demands associated with a fast growth rate or to
adapt to otherwise hostile environments, such as ones
where large amounts of double-stranded DNA breaks are
produced. This prediction may turn out to be true in Salmonella typhimurium, where strains deficient for RecBCD
lose their virulence in animals and tissue culture as a
result of their inability to resist the oxidative burst of
830 F. Chédin and S. C. Kowalczykowski
macrophages (Buchmeier et al., 1993, 1995; Spek et al.,
2001), a well-known source of DNA strand breaks
(Schlosser-Silverman et al., 2000).
In this light, it is interesting to note that there are
numerous cases of bacteria for which no clear homologue
of either RecBCD or AddAB enzyme can be detected.
Among the fully sequenced genomes presently available,
this situation applies for Aquifex aeolicus, Helicobacter
pylori, Mycoplasma genitalium, Mycoplasma pneumoniae, Rickettsia prowazekii, Deinococcus radiodurans,
Synechocystis and Treponema pallidum. However, most
of these organisms (with the exception of the mycoplasmas which possess the smallest bacterial genomes
known to date) encode homologues of the RecF and
RecR proteins, suggesting that the RecF pathway of
recombination has been well conserved throughout
evolution. It would be interesting to document whether
the species deprived of a functional RecBCD pathway are
more limited in their ability to adapt to new environments,
especially if these are associated with a high incidence of
DNA damage, and whether this handicap is compensated
by a higher mutability.
For species that contain a functional RecBCD or AddAB
homologue, however, the need for a regulatory circuit able
to turn off the voracious exonuclease activity is obvious.
Some bacteriophages have evolved systems to do just
that. The ends of the E. coli N15 phage are protected from
the RecBCD enzyme by the formation of a covalently
closed hairpin structure (Rybchin and Svarchevsky,
1999), whereas the T4 phage makes use of the endcapping gene 2 protein (Oliver and Goldberg, 1977). The
lambda phage utilizes the Gam protein, which directly
inhibits the activity of RecBCD (Murphy, 1991), while the
P22 phage modifies, to its advantage, the activity of the
RecBCD complex (Murphy, 2000). Bacteria, instead,
make use of regulatory sequences present in their
genomes. One argument frequently put forward to
rationalize this choice is the ‘password’ paradigm. c
sequences are usually over-represented in their respective genome (El Karoui et al., 1999), which could provide
their hosts with a password they can use to discriminate
self from non-self (Biswas et al., 1995; Cheng and Smith,
1984). It is worth mentioning, however, that this c-based
defence system has strong shortcomings: in the event
that a piece of foreign DNA possesses the host’s c
sequence, this DNA will be targeted for homologous
recombination and effectively incorporated into the
genome, a property that has actually been used to
achieve gene replacement in E. coli (Dabert and Smith,
1997). The ‘password’ paradigm can therefore only
operate on relatively short foreign DNA substrates or in
cases where the incoming DNA has no homologous locus
in the genome of the receiving bacterium. In the case
of conjugation, where exchanges can involve up to
megabases of DNA, this protection on its own will, most
likely, be ineffective. In the case of attempted interspecies
conjugation, the main enforcer for the ‘purity’ of the
exchange is the methyl-directed mismatch repair system
(Rayssiguier et al., 1989; Matic et al., 1995). The bacterial MutHLS system can indeed edit heteroduplex DNA
formed by DNA recombination and promote its reversal or
its destruction if mismatches are found (Stambuk and
Radman, 1998). This role that has been preserved
throughout evolution, since the mismatch repair system
also serves to prevent DNA recombination between
slightly divergent sequences in eukaryotes (Chen and
Jinks-Robertson, 1998; de Wind et al., 1995). A more
compelling case for the use of c sequences as a means
to regulate enzymes such as RecBCD or AddAB is found
in the co-ordination provided by this regulation. While all
the stratagems found in bacteriophages aim at permanently disabling the function of the enzyme, the use of a
regulatory sequence allows the cells to switch off the
nuclease function, while switching on the initiation of DNA
recombination in a process that can be repeated as many
times as necessary.
The usefulness of this regulatory switch has been
underlined in recent studies of the repair of spontaneous
dsDNA breaks in E. coli. Contrary to popular belief, a considerable number of dsDNA breaks occurs during each
replication cycle as a consequence of the interruption and
collapse of DNA replication forks in E. coli (Kuzminov,
1995; Kogoma, 1997; Michel et al., 1997; Kowalczykowski, 2000). The RecBCD enzyme, with its high
affinity for DNA ends and its unique ability to initiate effectively the recombinational repair of these lesions, controls
the main route by which cells can deal with this burden
(Seigneur et al., 1998). This explanation also helps to
rationalize the cost associated with the domestication of
RecBCD enzyme’s nuclease activity via regulation by c:
the over-representation of c sites in the genome and their
predominant co-orientation with the direction of DNA replication makes this repair even more effective. It is likely
that this explanation holds for other bacteria, including B.
subtilis and L. lactis. Indeed, their respective c sequences
are over-represented in their genomes and, at least for B.
subtilis and L. lactis, co-oriented with DNA replication (El
Karoui et al., 1999). It therefore appears that regulated
helicases/nucleases, rather than being merely domesticated machines of war, provide cells with exquisitely
efficient ways to cope with a particularly lethal type of
lesion such as dsDNA breaks.
Altogether, these data show that the E. coli RecBCD
enzyme and the B. subtilis AddAB enzyme constitute
two distinct, yet related, families of regulated helicases/
nucleases. Uncovering the functions of these enzymes,
and, in particular, elucidating the mechanism by which
their recombinogenic potential is regulated in response to
© 2002 Blackwell Science Ltd, Molecular Microbiology, 43, 823–834
A novel family of regulated helicases/nucleases
specific sequences, will undoubtedly offer some fascinating new insights into our understanding of the process of
DNA recombination in Bacteria.
Acknowledgements
We thank the following members of the Kowalczykowski
laboratory for their critical reading of the manuscript: Carol
Barnes, Cynthia Haseltine, Noriko Kantake, Julie Kleiman,
Jim New, Erica Seitz and Maria Spies. This work was supported by funds from the National Institutes of Health grant
GM-41347.
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