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Journal of Bacteriology, July 2001, p. 4071-4078, Vol. 183, No. 13
Laboratoire de Génétique
Appliquée1 and Collection
CNRZ
Received 16 January 2001/Accepted 11 April 2001
In bacteria, double-strand DNA break (DSB) repair involves an
exonuclease/helicase (exo/hel) and a short regulatory DNA sequence (Chi) that attenuates exonuclease activity and stimulates DNA repair.
Despite their key role in cell survival, these DSB repair components
show surprisingly little conservation. The best-studied exo/hel, RecBCD
of Escherichia coli, is composed of three subunits. In
contrast, RexAB of Lactococcus lactis and exo/hel
enzymes of other low-guanine-plus-cytosine branch gram-positive
bacteria contain two subunits. We report that RexAB functions via a
novel mechanism compared to that of the RecBCD model. Two potential nuclease motifs are present in RexAB compared with a single nuclease in
RecBCD. Site-specific mutagenesis of the RexA nuclease motif abolished
all nuclease activity. In contrast, the RexB nuclease motif mutants
displayed strongly reduced nuclease activity but maintained Chi
recognition and had a Chi-stimulated hyperrecombination phenotype. The
distinct phenotypes resulting from RexA or RexB nuclease inactivation
lead us to suggest that each of the identified active nuclease sites in
RexAB is involved in the degradation of one DNA strand. In RecBCD, the
single RecB nuclease degrades both DNA strands and is presumably
positioned by RecD. The presence of two nucleases would suggest that
this RecD function is dispensable in RexAB.
In bacteria, double-stranded DNA
breaks (DSB) are frequent events that may be provoked, for example, by
pauses in the replication fork (36, 43). Such genomic
disruptions are lethal in the absence of DNA repair. In
Escherichia coli DSB repair requires the activity of a large
enzyme complex, known as RecBCD, that has ATP-dependent helicase and
exonuclease activities (see reference 32 for a review).
The enzyme degrades both strands, starting from the DNA break until it
reaches an octanucleotide sequence, known as Chi, that attenuates
degradation and stimulates recombination (44, 46). The
enzyme exhibits helicase activity and residual exonuclease activity
with an altered polarity after Chi (4, 16); the remaining
activity provides a single-stranded DNA substrate for recombination
enzymes to mediate repair.
Organization of the three-subunit exonuclease/helicase (exo/hel)
RecBCD.
Structure-functional studies of RecBCD have revealed some
of the roles of each subunit. RecB seems to possess two key activities of the enzyme. The N-terminal 929 amino acids (out of 1,180 total) have
confirmed ATPase and helicase activities (13, 54); this region is similar to that of UvrD helicase. RecBCD helicase activity was recently proposed to function via a mechanism similar to that determined for UvrD (6). Nuclease activity was recently
localized to the C-terminal 251 amino acids of RecB and is associated
with the presence of a conserved motif, G-i-i-D-x(12)-D-Y-K-t-d
(amino acids in small letters show less conservation) (51, 53,
54). This motif is present in numerous bacterial and eukaryotic
enzymes (5). RecBCD was shown to have a single nuclease
catalytic center in RecB that works on both DNA strands
(51). Little is known about the roles of RecC, except that
it appears to greatly enhance activities and processivity of RecB
(11, 38); mutations in the RecC gene can also result in
loss or modification of Chi recognition, as do mutations in genes of
all subunits (1). RecD is an ATPase with similarity to a
helicase involved in conjugational transfer of an enteric bacterial
plasmid; its homologues seem to be broadly distributed in bacteria
(determined by BLAST comparisons; http://www.ncbi.nlm.nih.gov/BLAST/). As part of RecBCD, RecD appears to regulate exonuclease activity. Recent data suggest that RecD maintains RecBCD incompetent for homologous recombination prior to Chi; at Chi, RecD is suggested to
undergo a conformational change that attenuates exonuclease activity
and stimulates recombination (2, 3, 12, 33, 48). A swing
model was proposed in which RecD assures proximity of the RecB nuclease
with both DNA strands prior to Chi and a repositioning of the nuclease
after Chi (51, 54).
Organization of the two-subunit exo/hel enzymes.
To date,
models of exo/hel activities are based on those of RecBCD. Numerous
RecBCD homologues have been identified in gram-negative enterobacteria and in the high-guanine-plus-cytosine-content
mycobacteria. However, the functional RecBCD analogue in
the low-guanine-plus-cytosine-content branch of gram-positive
bacteria is structurally distinct from RecBCD. Using Lactococcus
lactis as a model, a two-subunit enzyme called RexAB (comprising
1,073- and 1,099-amino- acid subunits, respectively) is necessary and
sufficient to confer exo/hel activity and interacts with the L. lactis Chi site (22). RexAB bears homologues in at
least six other gram-positive low-guanine-plus-cytosine-content bacteria as well as in the gram-negative bacterium
Porphyromonas gingivalis (determined by BLAST
comparison;
http://www.ncbi.nlm.nih.gov/BLAST/). As studied
in L. lactis or in Bacillus subtilis (AddAB), the
two-subunit exo/hel enzymes display biological and/or biochemical
activities equivalent to those of RecBCD (i.e., ATP-dependent
exonuclease, helicase, exonuclease blocking at Chi, and Chi-stimulated
recombination; 8, 9, 10, 22, 23, 28, 30, 31). RexA and its analogues in other gram-positive bacteria have homology with PcrA helicase (determined by BLAST comparison;
http://www.ncbi.nlm.nih.gov/BLAST/), whose
mechanism has been deduced from structural determinations (49). In addition, the nuclease motif described above for
RecB is conserved in L. lactis RexA and in all known
two-subunit exo/hel enzymes (5, 28, 53). This similarity
has lead to the hypothesis that all exo/hel enzymes function via
similar mechanisms.
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.13.4071-4078.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
In Vivo Evidence for Two Active Nuclease Motifs in
the Double-Strand Break Repair Enzyme RexAB of
Lactococcus lactis
URLGA,2 Institut National de la Recherche
Agronomique, Domaine de Vilvert, 78352 Jouy en Josas, France
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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MATERIALS AND METHODS |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Strains and plasmids.
The E. coli strains used
were TG1 [F' traD36 lacIq
(lacZ)M15
proA+B+/supE
(hsdM-mcrB)5(rK
mK mcrB) thi
(lac-proAB)], AB1157 (argE3
his-4 leuB6 proA2
thr-1 ara-14
galK2 lacY1 mtl-1
xyl-5 thi-1
rpsL31 tsx-33 supE44), and KM21 (AB1157 isogenic strain containing [recC ptr recB
recD]::kan, referred to here as recBCD)
(37). The rexAB genes were derived from
L. lactis strain MG1363 (25) and were cloned on
low-copy-number plasmid pGB2 (confers spectinomycin resistance) to
generate pRexAB (22).
Media. E. coli strains were grown in Luria broth at 30 or 34°C, as specified below. Antibiotics were used in E. coli as follows: ampicillin at 100 µg/ml, spectinomycin at 50 µg/ml, tetracycline at 15 µg/ml, kanamycin at 40 µg/ml, and chloramphenicol at 15 µg/ml.
rexAB mutant constructions.
To generate
mutants affected in the nuclease motifs of RexAB, fragment mutagenesis
was performed using modified primers and pRexAB as template DNA.
RexABD910A was constructed by replacing the
SfcI-ClaI fragment that contains the 3' end of
rexB with a corresponding PCR fragment that was modified by
a point mutation. The SfcI-end primer was
5'-CTTTCTACAGATTACTTAGGGGCGATTGCGTATA-3' (the SfcI site is underlined; the GAC aspartate codon,
RexB position 910, is replaced by the GCG alanine codon;
changes are in bold). The ClaI-end primer was
5'-TCGACAAATCGATTTGAGAGGACAATATCGACA-3' (the
ClaI site is underlined). The resulting fragment was first subcloned onto an intermediate vector and then was cloned to replace the wild-type segment in pRexAB. The
RexAB
DYK mutant was
constructed essentially in the same way except that the
SfcI-end primer was
5'-CCAACTTTCTACAGATTACTTAGGGGCGATT//TCAAGTGCTCATTCATT-3' (the 9-codon deletion, RexB amino acid positions 910 to 912, is indicated by double slashes).
DYKB mutant was
constructed by two-step mutagenesis using four primers. A
rexAB PstI-EcoRI fragment can be
generated using two outside primers: A,
5'-GAAGTTCAACCAGTCAGTGAGTTTGTTCG-3' (the PstI
site present in rexA is 46 nucleotides downstream of this
primer), and B,
5'-GGGAATTCGGTACCATTGTTCTTCCTCCCTAACAGC-3' (the PCR-amplified fragment contains an added EcoRI
site [underlined] at the end of the rexA gene). To
generate an internal DYK codon deletion, overlapping oligonucleotides
that prime in opposite directions were designed: C,
5'-CACATTTGTAAATCTGTCCGT//AAATAATATAATCTTGTCAGC-3' (the
9-codon RexA deletion, positions 1114 to 1116, is indicated by double
slashes; this oligonucleotide generates a PCR fragment when coupled
with primer A), and D,
5'-GACAAGATTATATTATTT//ACGGACAGATTTACAAATGTG-3' (the 9-codon
RexA deletion, positions 1114 to 1116, is indicated by double slashes;
this oligonucleotide generates a PCR fragment when coupled with primer
B). To generate a PstI-EcoRI fragment in which
the DYK tricodon is missing, separate PCRs were first performed using
primers A plus C and B plus D. The template was pRexAB. These fragments
were purified, combined, and used as templates in a PCR containing
oligonucleotides A plus B. The resulting band of the expected size was
purified and cloned, and the PstI-EcoRI fragment
was finally recloned into PstI-EcoRI-cut pRexAB.
The resulting pRexADYKB
clone was confirmed by sequencing.
The plasmid
pRexAB771-1063 was
constructed by digesting pRexAB with ClaI-ScaI
filling in and religation. This resulted in an in-frame deletion in
rexB.
UV sensitivity tests for E. coli.
The
E. coli recBCD strains containing mutated or
wild-type rexAB alleles were maintained at 30°C. Note that
exonuclease activity of RexAB is thermosensitive in E. coli,
possibly reflecting an optimal growth temperature of L. lactis of 30°C. Tests to determine UV resistance were performed
as described previously (18).
T4g2 test for exonuclease activity in E.
coli.
The bacteriophage T4g2 amber mutant
(kindly provided by W. Wackernagel, University of Oldenburg,
Oldenburg, Germany) was used to evaluate exonuclease activity.
The gene 2 product encodes a protein which protects phage DNA
extremities from RecBCD degradation (34). The phage stock
was prepared on a recBC strain not containing a
supE mutation, so DNA ends of this phage mutant are
exonuclease sensitive. Therefore, the number of PFU is low when this
phage is titrated on strains expressing RecBCD. Lactococcal
exonuclease activity expressed from pRexAB wild-type and mutated
alleles was evaluated in the E. coli recBCD
strain and compared to that of plasmid-free AB1157 (wild type) and
E. coli recBCD strains essentially as
described (52), except that cultures and plates were
incubated at 30°C.
Detection of HMW. High-molecular-weight linear plasmid multimer (HMW) accumulation was detected on whole-cell lysates after agarose gel electrophoresis (14). Plasmid DNA, labeled by chemiluminescence using the ECL system (Amersham), was used as a probe. Southern blot hybridization was performed as recommended by the kit supplier.
Recombination test.
We used a previously described strategy
to measure Chi-stimulated homologous recombination using short dsDNA
substrates (15). The plasmid target (named pBla) is a
pBR322 derivative with a 111-bp deletion in the 
-lactamase gene
(bla). The intact bla gene is restored via a
double-exchange event with a linear DNA fragment (see reference
22 for details of its construction). In brief, primers
were designed to PCR amplify a bla gene internal fragment
covering the DNA deleted from pBla plus an additional 360-bp
flanking homology with bla. Primer couples generating double ChiLl sites or no Chi sites
(ChiLl0) were as described previously
(22). ChiLl sites are located about 300 bp
from heterologous dsDNA ends. Linear DNA used for experiments was
recovered by PCR using the primers
5'-GTTGGGAAGGGCGATCGGTG-3' and
5'-CACTCATTAGGCACCCCAGGC-3'. The final fragment
sizes were ~1.3 kb.
recBCD strain with or
without plasmids encoding the different RexAB alleles plus pBla were prepared at 34°C, and control strain TG1 carrying pBla was
prepared at 37°C. Cells were incubated at 34°C for 90 min after
electrotransformation, and colony counts were determined after a 2-day
incubation. Competence was determined by transforming cells with known
amounts of pACYC184 DNA, selecting for chloramphenicol. Strains were
transformed with about 200 ng of linear DNA as described previously
(17). Linear DNA samples were quantitated on agarose gels
using marker DNAs of known quantities. Electrotransformation into TG1
carrying pBla was used to evaluate DNA quality. It was previously
shown that electroporation totally inactivates wild-type E. coli RecBCD exonuclease but allows E. coli
Chi-independent recombination (20); the recombination capacity of the two fragments (regardless of
ChiLl) was compared in this way. Although the
reason why electroporation inactivates RecBCD exonuclease is unknown,
it is possible that the electric shock induces the SOS response, which
is known to diminish exonuclease activity and retain recombination
proficiency (41). Both fragments were found to transform
this strain with about equal efficiencies (see Table 2).
Colony counts were performed after 48 h of incubation. The numbers
of Amp-resistant transformants obtained with
ChiLl- or ChiLl0-containing linear DNAs
were compared for each strain. Linear DNA samples were quantitated on
agarose gels using marker DNAs of known quantities. Electrocompetent
E. coli recBCD containing pBla was used as
a negative recipient control.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Conserved regions of two- versus three-subunit exo/hel
enzymes.
Alignments of several two-subunit exo/hel enzymes reveal
that these enzymes are poorly homologous, even in closely related species. They bear very little homology with the three-subunit exo/hel.
Nevertheless, a short nuclease motif was previously revealed in the
AddA subunit of the B. subtilis AddAB enzyme and in the RecB
subunit of RecBCD (28, 53). In RecBCD, this motif
corresponds to the sole nuclease activity of the exo/hel enzymes
(51). We found that this motif is actually present in both
subunits of the two-subunit enzymes (Fig.
1). In the RexA subunit, a consensus is
G-i-i-D-x(12)-D-Y-K-t-d (amino acids in small letters show some
variation); in RexB it is G-r-i-D-R-i-D-x(9-12)-v-D-Y-K-S-s. The
striking similarities between these motifs lead us to ask whether RexAB
may bear two active nuclease sites, one in each subunit.
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Site-specific mutagenesis of putative nuclease motifs in RexA and
RexB.
The rexAB operon (rexB is followed by
rexA) was previously cloned on a low-copy-number plasmid and
shown to fulfill the biological roles of RecBCD in an E. coli recBCD strain (22). The putative RexA nuclease motif (RexANuc) was modified by
a 3-amino-acid deletion removing the tripeptide DYK in positions 1114 to 1116 (called
RexADYKB) (Fig. 1). The
putative RexB nuclease motif (RexBNuc) was
modified by alteration of D910 to A (called
RexABD910A) or by a 3-amino-acid deletion,
removing the tripeptide DYK in positions 910 to 912 (called
RexABDYK) (Fig. 1). In
addition, a 293-amino-acid in-frame deletion of RexB that removed
C-terminal amino acids 771 to 1063 (called
RexAB771-1063) was
constructed. The cloned rexAB mutated genes gave rise to pRex plasmids bearing the name of the mutation and were established in
an E. coli recBCD strain (37).
recBCD
strain shows greater UV resistance in the presence of pRexAB
(22) or pRexABD910A. However,
UV resistance of the recBCD strain was not at all or
was only very slightly improved in the presence of
pRexADYKB,
pRexAB771-1063, or
pRexABDYK compared to
that of the control strains. These results indicate that the introduced
mutations affect the DNA repair capacity of RexAB exo/hel.
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Changes in RexA or RexB nuclease motifs affect RexAB exonuclease
activities.
Phage T4g2 is susceptible to exo/hel
degradation. As nuclease activity inhibits plaque-forming ability,
plaque formation would reflect diminished nuclease activity in the
rexAB mutants (Table 1). The recBCD mutant
containing pRexAB efficiently inhibits phage multiplication, whereas
this strain lacking rexAB genes is totally permissive
(22). The strain containing
pRexADYKB allowed
efficient phage multiplication. Thus, inactivation of the nuclease
motif of RexA essentially abolishes all DNA degradation activity by RexAB.
DYK,
T4g2 infectivity was increased 10- and 100-fold,
respectively (Table 2), indicating that
nuclease activity is significantly reduced in these strains. Note that
nuclease inactivation may even be underestimated in this assay, as
unwinding activity alone may have a modest inhibitory effect on
T4g2 multiplication (40). This result shows
that the RexB nuclease motif DYK is a functionally active component of the exo/hel enzyme. The strain containing
pRexAB771-1063 is
totally permissive for phage multiplication, indicating that a large
deletion in RexB abolishes in vivo nuclease activity.
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RexBNuc mutants recognize Chi.
We previously
developed an in vivo test to detect Chi attenuation of exo/hel
exonuclease activity by using an RC plasmid as substrate. A 
-formed
replication intermediate of RC plasmids provides a dsDNA end as an
entry point for exo/hel enzyme. If the RC plasmid contains a Chi site
in the orientation recognized by exo/hel, degradation is attenuated and
-form replication results in accumulation of HMW (14,
26). In the absence of Chi on the plasmid, HMW do not accumulate
as long as exo/hel is active. However, if exo/hel is nuclease
defective, any RC plasmid will accumulate HMW (e.g., RC plasmids
accumulate large amounts of HMW in an E. coli recD mutant;
our unpublished data).
recBCD background on RC plasmids with or
without an L. lactis Chi site (called
ChiLl) (Fig. 2). In
the presence of pRexAB, HMW accumulation was observed only if
ChiLl was present on the RC plasmid. In the absence of any exo/hel enzyme, HMW accumulated regardless of the presence of ChiLl on the plasmid (Fig. 2)
(22). The strain containing pRexADYKB behaved like
the exo/hel-deficient strain. These results are consistent with the
nuclease-negative phenotype conferred by
RexADYKB in the phage
infection test. Other mutated rexA alleles in which the
nuclease motif was deleted gave rise to similar phenotypes (L. Rezaïki and A. Gruss, unpublished observations). We also observed ChiLl-independent HMW accumulation in
the strain expressing RexAB771-1063,
confirming that nuclease activity is deficient in this enzyme.
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DYK
was present; a modest effect of ChiLl in
increasing accumulation was still observed. These results are
consistent with our hypothesis that the RexBNuc
motif is necessary for nuclease activity of the enzyme. In addition, the ChiLl-dependent increase in HMW accumulation
shows that the remaining nuclease activity is still attenuated at
ChiLl. Possibly, the RexB subunit degrades just
one of the two dsDNA strands from the 5' end (i.e., the strand
containing the Chi complement) (23).
These results confirm the nuclease-defective phenotype of
RexBNuc as seen in the T4g2 test. They
further show that although RexBNuc nuclease
activity is reduced, its ChiLl recognition is
maintained. In contrast, the RexANuc nuclease
change abolishes all nuclease activity, regardless of ChiLl. Thus, the phenotypes of the RexA and RexB
nuclease changes are clearly distinguishable in vivo.
RexBNuc but not RexANuc mutants display a
Chi-stimulated hyperrecombination phenotype.
Gene replacement with
linear DNA fragments is stimulated if correctly oriented Chi sites are
present in the linear DNA flanking regions of homologies (15, 22,
23). Using this criterion, it was previously demonstrated that
the presence of ChiLl stimulates homologous
recombination (22, 23). We examined the ability of mutant
RexAB exo/hel to mediate ChiLl-stimulated
homologous recombination by using linear fragments with or without
double ChiLl sites at the ends (Fig.
3 and Table 2). The E. coli
recBCD recipient contained the recombination target
plasmid (pBla) together with a plasmid encoding a mutated
rexAB allele. As expected, very few recombinants were
obtained in the recBCD host, regardless of whether
ChiLl were present on the linear dsDNA ends.
Recombination via wild-type RexAB was stimulated 30-fold by the
presence of Chi on incoming linear fragments, in keeping with previous
results (22, 23). In the presence of
pRexADYKB, recombination
was at background levels, further confirming that a change in the RexA
nuclease motif results in total inactivation of RexAB biological
activities in vivo.
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DYK. Thus,
RexBNuc mutants have a stimulatory effect on
recombination using short DNA fragments as substrates, and they also
retain Chi recognition.
The strain containing
pRexAB771-1063
demonstrated recombination frequencies like those of pRexAB, except
that ChiLl0 fragment frequencies were
elevated; nevertheless, an approximately threefold Chi stimulation
effect was observed. These results suggest that the
pRexAB771-1063 enzyme
retains some ChiLl recognition activity despite a
large C-terminal RexB deletion.
Taken together, these results show that RexA and RexB subunits both
contribute to the observed nuclease activity of RexAB. The
RexADYK mutant abolishes
all detectable in vivo activity of the enzyme, including homologous
recombination. The RexABD910A and
RexBDYK mutants reduce
exonuclease activity but retain ChiLl recognition and display a ChiLl-stimulated hyperrecombination
phenotype when using short dsDNA fragments as substrates.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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RexAB exo/hel enzyme function involves two active nuclease sites. The E. coli RecBCD-Chi couple has served as the prototype for bacterial DSB repair. Indeed, exo/hel-Chi couples identified in other bacteria were confirmed to fulfill the biological or biochemical functions established in E. coli (9, 22, 42, 52). However, RecBCD and the L. lactis exo/hel enzyme, RexAB, differ operationally: RecBCD relies on a single nuclease to effect DNA degradation (51, 53). In contrast, we have shown that RexAB contains two nuclease motifs, one in each subunit, both of which are required for full nuclease activity. Inactivation of the RexA nuclease motif results in total loss of exo/hel functions in vivo, whereas inactivation of the RexB nuclease motif reduces degradation while retaining Chi activity. As these two nuclease motifs are present in all identified (or predicted) two-subunit exo/hel enzymes, we predict that these enzymes will have properties similar to those described here.
We propose a model for RexAB activity based on our in vivo results (Fig. 4A). The two major features of the model are the following. (i) RexA, like RecB, has helicase and nuclease activities and drives the enzyme. Unwinding of the double helix is assured by RexA, which has significant homology with PcrA helicases. The RexB subunit could enhance activities of RexA helicase, just as RecC appears to increase RecB processivity and activities (11, 38). (ii) The RexA nuclease cleaves just one strand, from the 3' end, while the RexB nuclease is positioned to degrade from a 5' end. This model is consistent with our results showing that RexBNuc mutants degrade DNA but maintain Chi recognition and with recent in vitro studies reported for AddAB in which degradation is attenuated at Chi but only on the Chi-containing strand; degradation of the "bottom" strand continues after Chi (9). This model may be useful in understanding the absence of a third, RecD-like component in the two-subunit exo/hel enzymes. RecD is proposed to position the single nuclease of the RecBCD enzyme so that it degrades the two DNA strands (51); if, as proposed, the nucleases of RexAB each act on a DNA strand, this RecD function would not be needed. Alternatively, the two established nuclease motifs are both needed to degrade each strand. We consider this possibility unlikely, as our results show that the RexA nuclease is active even if RexBNuc is mutated. In vitro analyses will confirm whether this model is valid.
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DYKB mutant
exhibited no in vivo biological activity and may thus correspond to a
null mutant. Interestingly, in B. subtilis and in E. coli, mutations affecting nuclease motifs in RexA analogues AddA
and RecB, respectively, retain helicase activities. For example, an
AddAB mutant in which the C-terminal end of AddA is deleted lacks
nuclease activity but retains some helicase proficiency in vivo and in
vitro (28), and an E. coli
RecBD1080ACD gene mutation inactivates the
RecB nuclease (53); in vivo, this enzyme is nonfunctional
(2). It was reasoned that
RecBD1080ACD is unable to undergo a
conformational change at Chi and thus remains locked in the
"antirecombinase" position; this effect is alleviated by removing
RecD (2, 12). Our preliminary results suggest that
recombination is restored in a
RexADYK
RexBDYK double mutant
(data not shown), suggesting that the conformational change observed at
ChiLl might involve interactions between RexB and
RexA nuclease domains. Applying the above reasoning, we speculate that
RexADYKB could be locked
in a nonrecombinogenic state that blocks the conformational change at
ChiLl needed to render it recombinogenic. The
alternative hypotheses concerning the RexANuc
mutant will be examined by further genetic and biochemical tests.
Why is exo/hel so poorly conserved? The primordial need for an intact genome suggests that DNA genome repair mechanisms have been present early in evolution. Accordingly, generalized homologous recombination proteins such as RecA, SSB, and RecF are rather highly conserved (19) (comparisons analyzed using BLAST; http://www.ncbi.nlm.nih.gov/BLAST/).
It is surprising that components of an important survival function like DNA repair are so markedly divergent. The diversity in DSB repair enzymes may be related to genome plasticity: genome rearrangements are common events that may occur via intrachromosomal transposition, gene duplications and inversions, or entry of exogenous DNA. In L. lactis, there is a 4:1 orientation bias of Chi distribution with respect to the direction of DNA replication (exo/hel recognizes Chi in one orientation) (14,21,23,29,47). Inversion of a large DNA segment could considerably reduce the number of Chi sites available to stimulate repair if a replication fork break occurs in that region (36). Such rearrangements could impose selective pressure for exo/hel divergence. The constant need for the exo/hel enzyme to adapt to altered distributions of Chi on the genome (e.g., due to chromosomal inversions or mutations) could explain why these enzymes are so divergent, even in closely related species. Is E. coli the right DSB repair model? The E. coli RecBCD exo/hel enzyme has been the paradigm for DSB repair over the last 30 years. But E. coli is a relatively young bacterium in terms of evolution; it seems to have evolved well after oxygen became abundant (27). In contrast, L. lactis, which thrives under low- or no-oxygen conditions, appears to have preceded E. coli evolutionarily (24, 27). To follow the evolution of the DSB repair system, we suggest that comparison with an older microorganism like L. lactis will be informative and may reveal minimum requirements for DSB repair.| |
ACKNOWLEDGMENTS |
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We appreciate discussions in the course of this work with Susan Amundsen, Yakyha Dieye, Bénédicte Michel, Yves Le Loir, Philippe Langella, and our laboratory colleagues and we thank anonymous reviewers for comments.
This work was supported by a grant from the Programme de recherche fondamentale en microbiologie et maladies infectieuses et parasitaires of the Ministère de l'Education Nationale, de la Recherche et de la Technologie, France. A.Q. was the recipient of a postdoctoral fellowship awarded by the Consejo Nacional de Investigaciones Cientificas y Tecnicas, Argentina.
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FOOTNOTES |
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* Corresponding author. Mailing address: Laboratoire de Génétique Appliquée, Institut National de la Recherche Agronomique, Domaine de Vilvert, 78352 Jouy en Josas, France. Phone: 33-1 34 65 21 68. Fax: 33-1 34 65 20 65. E-mail: gruss{at}biotec.jouy.inra.fr.
Permanent address: Facultad de Ingenieria Quimica, Universidad
Nacional del Litoral, Santiago del Estero 2829, 3000 Santa Fe, Argentina.
Permanent address: National Center for Cell Science, Ganeshkhind,
Pune 411007, India.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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| 1. | Amundsen, S. K., A. M. Neiman, S. M. Thibodeaux, and G. R. Smith. 1990. Genetic dissection of the biochemical activities of RecBCD enzyme. Genetics 126:25-40[Abstract]. |
| 2. |
Amundsen, S. K.,
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Journal of Bacteriology, July 2001, p. 4071-4078, Vol. 183, No. 13
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.13.4071-4078.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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