By its functional interaction with a RecA polymer, the mutagenic
UmuD'C complex possesses an antirecombination activity. We show
here that MucA'B, a functional homolog of the UmuD'C complex, inhibits homologous recombination as well. In
F
recipients expressing MucA'B from a
Ptac promoter, Hfr × F
recombination decreased with increasing MucA'B concentrations down to 50-fold. In damage-induced pKM101-containing cells expressing MucA'B from the native promoter, recombination between a UV-damaged F lac plasmid and homologous chromosomal DNA decreased
10-fold. Overexpression of MucA'B together with UmuD'C resulted in
a synergistic inhibition of recombination. RecA[UmuR] proteins, which
are resistant to UmuD'C inhibition of recombination, are inhibited by
MucA'B while promoting MucA'B-promoted mutagenesis efficiently.
The data suggest that MucA'B and UmuD'C contact a RecA polymer at
distinct sites. The MucA'B complex was more active than UmuD'C in
promoting UV mutagenesis, yet it did not inhibit recombination more
than UmuD'C does. The enhanced mutagenic potential of MucA'B may
result from its inherent superior capacity to assist DNA polymerase in trans-lesion synthesis. In the course of this work, we
found that the natural plasmid pKM101 expresses around 45,000 MucA
and 13,000 MucB molecules per lexA(Def) cell devoid of
LexA. These molecular Muc concentrations are far above those of the
chromosomally encoded Umu counterparts. Plasmid pKM101 belongs to a
family of broad-host-range conjugative plasmids. The elevated levels of
the Muc proteins might be required for successful installation of
pKM101-like plasmids into a variety of host cells.
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INTRODUCTION |
SOS mutagenesis results from
inaccurate trans-lesion DNA synthesis carried out by a DNA
polymerase, probably DNA polymerase III, in conjunction with RecA and a
mutagenic complex formed by the two SOS proteins UmuD' and UmuC
(for a review, see reference 44). The UmuD'C
complex is thought to assist DNA polymerase in DNA synthesis
across a lesion, while a RecA polymer acts as a directional chaperone
to position the UmuD'C complex right at a lesion (6, 11,
38). A RecA polymer also acts as a recombinase in homologous
recombination (for a review, see reference 22) and
as a coprotease in the cleavage of LexA repressor and UmuD, the native
form of the mutagenically active UmuD' (for reviews, see
references 21 and 44).
In addition to being an essential component of trans-lesion
DNA synthesis, the UmuD'C complex is an antagonist of RecA-mediated recombination. When prematurely expressed at elevated intracellular levels, the UmuD'C complex prevents recombinational repair of UV-damaged DNA as well as recombination of undamaged DNA
(37). The antirecombination activity of the UmuD'C complex
correlates with its interaction with a RecA polymer (6, 38).
Here, we sought to determine if the property of the UmuD'C
complex of inhibiting homologous recombination is shared by other mutagenic complexes. We tested the MucA'B complex, which is a member of a family of mutagenesis proteins structurally and
functionally related to the UmuD'C complex (23, 46).
The MucA'B complex originates from plasmid pKM101, a variant of the
multidrug resistance plasmid R46 (27). Like the chromosomal umuDC genes, the mucAB genes are organized in
an operon and are regulated by LexA (12, 29). Like UmuD,
the native MucA protein is processed into an active form,
MucA' (17, 35). Among the mutagenic repair genes,
the mucAB genes from pKM101 appear to be the
most efficient in promoting mutagenesis (5, 24). This property has led to their inclusion in the Ames tester strains, which
detect environmental mutagens (25).
We found that the MucA'B complex inhibits homologous recombination
as does its cellular UmuD'C counterpart. In F recipients
expressing MucA'B from a Ptac promoter,
conjugational recombination decreased with increasing MucA'B
concentrations down to 50-fold. In damage-induced pKM101-containing
cells expressing MucA'B from the native promoter, recombination
between a UV-damaged F lac plasmid and homologous
chromosomal DNA also decreased markedly. Overexpression of MucA'B
with UmuD'C results in a synergistic inhibition of recombination.
Interestingly, the mutant RecA[UmuR] proteins, which are
resistant to UmuD'C (38), remain sensitive to MucA'B.
These data suggest that MucA'B and UmuD'C interact with a RecA
polymer at distinct sites.
| |
MATERIALS AND METHODS |
Bacterial strains, plasmids, culture, and media.
The
bacterial strains and plasmids used are described in Tables
1 and 2,
respectively. Plasmid pGY9411 was constructed by inserting the
mucA'B NcoI-BamHI fragment of pRW294 into the
vector pTrc99A under control of the Ptac
promoter and the associated Shine-Dalgarno sequence. Plasmid
pGY9463 (mucA') was derived from pGY9411 by truncating
the mucA'B operon at the BglII site, thus deleting 95% of mucB. Plasmid pGY9863 was derived from
plasmid pGY9738 (o1cumuD'C) by
replacing the mutant o1c with the
wild-type operator. The three constructs were verified by sequencing
through the modified regions.
Bacteria were grown at 37°C. The growth of the cultures was monitored
by absorbance at 650 nm. Minimal medium was M9 supplemented with 0.2%
(wt/vol) glucose, 30 µg of thiamine per ml, and the required amino
acids. The rich media LBT and LAT, enriched minimal medium M9C, and
low-arginine solid medium were as described previously (3,
6). Bacteria containing plasmids were grown in media supplemented
with the appropriate antibiotics. Antibiotics and chemicals were used
at the following final concentrations: streptomycin, 200 µg/ml;
kanamycin, 50 µg/ml; tetracycline 10 µg/ml; ampicillin, 15 µg/ml
for pKM101 or 100 µg/ml for derivatives of pTrc99A; chloramphenicol, 10 µg/ml; spectinomycin, 100 µg/ml for chromosomal spectinomycin resistance (Spcr) mutations or 50 µg/ml for derivatives
of pGB2; rifampin, 100 µg/ml;
5-bromo-4-chloro-5-indolyl-
-D-galactoside (X-Gal), 40 µg/ml; and isopropyl--D-thiogalactopyranoside (IPTG),
0.5 mM if not otherwise stated.
Conjugational crosses.
As a rule, donor and recipient
bacteria were grown in LBT to 2 × 108
ml1 and mated on a filter at a 1.5:1 donor/recipient
ratio. After 60 min, the mating mixture was vigorously shaken in M9,
and diluted samples were plated on selective plates.
In conjugation experiments in which zygotic induction of prophage 
was determined, HfrH (ind553) donors and F
recipients were mated in liquid at a 1:10 donor/recipient ratio for 40 min. Phages resulting from zygotic induction were plated on a lawn of
GY8500 (F+) indicator bacteria (6).
Rescue of a chromosomal lac marker by a UV-damaged F42-10
plasmid was measured by using a double mating procedure
(16). The UV-damaged F42-10 plasmid was first transferred to
an F recA+ recipient, where
recombination took place. The surviving plasmids were then transferred
to a second indicator recipient that was recombination deficient and
had the lac region deleted to determine their lac genotype.
Assay of UV mutagenesis.
Derivatives of AB1157
[argE3(Oc) Rifs] were grown in enriched
minimal medium M9C to an optical density at 650 nm (OD650)
of 0.3, centrifuged, resuspended in M9 buffer, and exposed to UV light.
To measure argE3
arg+ mutagenesis, the
UV-irradiated cells were plated on minimal medium containing a trace
amount of arginine to select for the induced Arg+
revertants and on minimal medium fully supplemented with arginine for
counting of the total number of surviving cells. To measure mutagenesis
to rifampin resistance (Rifr), the UV-irradiated cells were
diluted fivefold in LBT medium, incubated overnight, and plated on LAT
plates with and without rifampin to determine the frequency of the
induced Rifr mutants.
Quantitation of MucA, MucA', and MucB.
Whole-cell extracts were prepared from a known amount of cells as
described by Nastri et al. (28). Cell concentrations were determined by counting bacteria in a Malassez chamber with a
microscope. Protein concentrations were measured with a protein assay
kit (Bio-Rad) by the manufacturer's microassay procedure. Portions of
the cell extracts were run on a sodium dodecyl sulfate-15% polyacrylamide gel along with increasing amounts of purified MucA, MucA', and MucB proteins (31) as standards. The
proteins were transferred to a nitrocellulose membrane and treated with
a 1:5,000 dilution of anti-MucA and -A' antibodies (17)
or with a 1:25,000 dilution of anti-MucB antibodies
(43). MucA, MucA', and MucB proteins were
visualized by using 125I-labeled protein A, and the
corresponding bands were scanned with a PhosphorImager and quantified
with Image-Quant software (Molecular Dynamics).
| |
RESULTS |
A low level of MucA' is expressed from plasmid
pRW294.
In an effort to construct a mutant constitutively
expressing the MucA'B complex, we found an unexpectedly low
steady-state level of MucA' in lexA(Def)
bacteria carrying plasmid pRW294, which carries an engineered
mucA'B operon (Table
3). In comparison, lexA(Def) bacteria carrying plasmid pRW144, a
corresponding mucAB plasmid, had a 15-fold higher
MucA concentration (11,000 MucA molecules versus 700 MucA'
molecules per cell) (Table 3). The steady-state level of MucB
expressed from pRW294 was also reduced compared to that from pRW144 but
not as dramatically (Table 3). MucA' was efficiently produced from
MucA in mitomycin-treated lexA(Def) cells,
suggesting that the difference between MucA and MucA'
expression (Table 3) was not due to a high rate of degradation of
MucA' by a cellular protease. We also checked by sequencing that
our isolate of plasmid pRW294 did not carry a mutation in the promoter
or the Shine-Dalgarno sequence that could account for the poor
MucA' expression. We suggest that the deletion of the first 26 N-terminal codons of mucA, giving rise to
mucA' (36), might cause an unfavorable change
in the mRNA secondary structure that might impair transcription or
translation of the engineered mucA'B operon.
Elevated levels of Muc proteins are expressed from plasmid
pKM101.
The data in Table 3 also show that
lexA(Def) bacteria carrying plasmid pKM101 had around
45,000 MucA, 48,000 MucA' (after mitomycin treatment), and
13,000 MucB molecules per cell. The concentration of RecA in the
same cells, which provided an internal standard, was approximately
76,000 monomers per cell, a value in agreement with previous
evaluations (6, 32).
The levels of the Muc proteins expressed from pKM101 appear to be
surprisingly elevated, considering the low-level production of their
chromosomally encoded Umu counterparts [2,500 UmuD and 200 UmuC
molecules per lexA(Def) cell (45)].
Interestingly, the Muc levels expressed from pKM101 were also
markedly higher than those expressed from plasmid pRW144, in which the
mucAB operon has been subcloned (Table 3). This suggests
that plasmid pKM101 may carry a regulatory element enhancing the
expression of the mucAB operon upon SOS induction. In
the leading region of pKM101 there are several conserved elements
possessing a strong promoter and an SOS box-like sequence
(10); however, none of these elements is in the
mucAB orientation so as to play a regulatory role in MucAB expression. Alternatively, expression of MucAB may be
sensitive to the level of supercoiling, which may differ between the
large pKM101 plasmid and the small pRW144 plasmid. Finally, it should be noted that the levels of the Muc proteins expressed from pKM101 in an SOS-induced cell are far above those required for efficient mutagenesis (see below). This raises the question whether a high concentration of the Muc proteins is required for an additional plasmid pKM101 function.
Enhanced mutagenic activity of the MucA'B complex.
The
poor expression of MucA'B from plasmid pRW294 nevertheless entailed
high levels of SOS mutagenesis. Indeed, 
umuDC bacteria carrying plasmid pRW294 were threefold more UV mutable than
corresponding cells carrying a umuD'C plasmid and sixfold
more UV mutable than wild-type umuDC+ bacteria
(Fig. 1). These results are in line with
those of Lawrence et al. (24) for single-stranded phage
mutagenesis and indicate that the MucA'B complex is mutagenically
more active than the UmuD'C complex. Thus, two factors appear to
contribute to the elevated mutability of mucAB bacteria:
speed of MucA processing (17) and efficient mutagenic
activity of MucA'B.
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FIG. 1.
The MucA'B complex is more mutagenic than UmuD'C.
Derivatives of GY9743 (umuDC) carrying plasmid pRW294
(mucA'B) (filled squares) or plasmid pGY9863
(umuD'C) (filled circles) and GY4587
(umuDC+) (open triangles) were assayed for
UV-induced Arg+ (left panel) or Rifr (right
panel) mutants after exposure to the UV doses indicated on the
abscissa.
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Inhibition of Hfr × F recombination as a
function of the MucA'B concentration in recipient bacteria.
We
wanted to assess whether the MucA'B complex behaves as an inhibitor
of homologous recombination. For this purpose, we measured Hfr × F recombination in recipient bacteria in which the
concentration of the MucA'B complex was made to vary by having the
mucA'B genes placed under the control of a
tac promoter. Expression of MucA'B from the hybrid
operon was induced with increasing IPTG concentrations and monitored by
measuring the levels of MucA' and MucB. The concentration of
the MucA'B complex was equated to the concentration of MucB that was produced in a limited amount relative to its MucA' partner (Table 4).
The efficiency of conjugational recombination decreased sharply when
the concentration of the MucA'B complex increased (Fig. 2). A basal level of MucA'B, around
350 MucA'B complexes per cell, reduced the yield of
recombinants twofold as compared to that of a corresponding
recipient devoid of MucA'B. When the MucA'B concentration
increased to around 1,600 complexes per cell, recombination decreased sixfold. A 10-fold-higher MucA'B concentration inhibited recombination more than 50-fold. However, a greater increase in the
MucA'B concentration did not reduce further the recombination efficiency. We checked that overexpression of the MucA'B complex did not prevent the transmission of the Hfr DNA by measuring zygotic induction of prophage in parallel crosses with a lysogenic
derivative of the HfrH donor (6) (data not shown).
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FIG. 2.
Inhibition of Hfr × F recombination
as a function of increasing MucA'B cell concentration. GY9427
(F leu::Tn10 umuDC) (open
symbol) and its derivative carrying plasmid pGY9411
(Ptac::mucA'B) (filled
symbols) were mated with GY306 (HfrH), and selection was for
Leu+ Strr recombinants. Prior to mating,
expression of MucA'B in the recipient bacteria was induced with
IPTG as described in Table 4, footnote a, so as to attain
the MucA'B cell concentrations indicated on the abscissa.
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Both the MucA' and MucB proteins are required to inhibit
homologous recombination. Indeed, there was no reduction
of recombination in the absence of MucB in
recipients producing MucA' alone (data not
shown). Furthermore, the native MucAB complex produced at an elevated concentration from plasmid pGW16, an
operator-constitutive derivative of pKM101 (26),
inhibited recombination only twofold (data not shown).
We conclude that the mutagenically active MucA'B complex, like the
UmuD'C complex, possesses an antirecombination activity; however, the
present results, compared with the data of Sommer et al.
(37) and of Boudsocq et al. (6), suggest that
MucA'B is rather less efficient than UmuD'C in inhibiting
homologous recombination. Indeed, compared to UmuD'C, a higher
concentration of the MucA'B complex is required for inhibiting
recombination. In our system, about 1,600 MucA'B complexes per cell
caused a sixfold drop in recombination, whereas a concentration of 700 UmuD'C complexes per cell induced from a PBAD
promoter or 440 UmuD'C complexes per cell constitutively expressed from a mutant operator produced a similar inhibition (6, 37).
pKM101-promoted inhibition of recombination between an
incoming UV-damaged F lac plasmid and homologous
chromosomal DNA in SOS-induced recipient cells.
Does the
MucA'B complex expressed from its natural promoter under
physiological conditions, i.e., in damage-induced pKM101-containing cells, inhibit homologous recombination? To answer this question, we
tested whether pKM101 would inhibit recombination between an incoming
UV-damaged F lac plasmid and homologous chromosomal DNA in
recipients that had been induced with mitomycin before the F conjugal transfer.
Recombination of the UV-damaged F lac plasmid occurred at a
high frequency in a recipient devoid of pKM101. Indeed, about 20% of
the surviving plasmids were found to have incorporated the chromosomal
lacZ::kan allele (Fig.
3, right panel).
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FIG. 3.
pKM101-promoted inhibition of recombination between an
incoming UV-damaged (UV'ed) F lac plasmid and homologous
chromosomal DNA in recipients induced with mitomycin. GY8630
(recA13 uvrB501/F42-10) donors exposed to 25 J of UV
light m2 were mated with GY10419 (F
lacZ::kan umuDC Strr) (right
panel) or its derivative carrying plasmid pKM101 (left panel)
pretreated for 90 min with 0 (open bars) or 2 (filled bars) µg of
mitomycin per ml. After this first mating, the exconjugants were mated
again with the indicator recipient GY7962 [F
recA99(Am) lac Spcr] and plated
on X-Gal plates supplemented with tetracycline and spectinomycin and
with or without kanamycin to determine the frequencies of
lacZ::kan recombinants as indicated on the
ordinate.
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An elevated recombination frequency was also observed in a recipient
carrying pKM101 in the absence of an SOS-inducing pretreatment (Fig. 3,
left panel). In contrast, when these cells were treated with mitomycin,
the yield of F lacZ::kan recombinants
decreased 10-fold (Fig. 3, left panel). This drop in recombination
is correlated with the enhanced production of the Muc
proteins, which were found to increase from very low basal levels
(below our detection limits) to around 8,000 MucA, 20,000 MucA', and 7,000 MucB molecules per cell after mitomycin
induction (Table 3). Furthermore, no recombination inhibition
occurred when pKM101 carried a
mucA421::Tn5 mutation (data not shown).
Conjugal transfer of UV-damaged single-stranded DNA generates
discontinuities (DNA gaps) in the complementary strand
synthesized in the recipient (19). Similar gaps arise after
replication of damaged chromosomal DNA (30). Our
results suggest that the MucA'B complex, when it reaches a
high enough concentration after DNA damage, inhibits the
recombinational filling of unresolved postreplicative gaps.
Synergism of MucA'B and UmuD'C in inhibiting
recombination.
The kinetics of recombination inhibition by
MucA'B shown in Fig. 2 indicates that the target sites become
saturated at high MucA'B concentrations. We asked whether
recombination could be further inhibited if MucA'B was expressed in
a recipient that also overproduces the UmuD'C complex. For this
purpose, the Ptac::mucA'B construct was introduced into GY9461, an
o1c-umuD'C recipient
constitutively expressing around 1,000 UmuD'C complexes per cell
(37) (data not shown). In the absence of MucA'B,
GY9461 bacteria showed a 30-fold decrease in recombination proficiency
(Fig. 4, right panel) (37).
However, when MucA'B was also expressed in the same cells,
recombination was further inhibited (Fig. 4, right panel). At a
MucA'B concentration of around 20,000 complexes per cell,
recombination was decreased more than 1,000-fold, while it decreased
only 50-fold in the absence of UmuD'C (Fig. 4, left panel).
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FIG. 4.
Synergistic inhibitory action of MucA'B and UmuD'C
on recombination. GY9427 (F
leu::Tn10 umuDC) (left panel) and GY9461
(F leu::Tn10
umuDC/o1cumuD'C) (right panel)
and their derivatives carrying plasmid pGY9411
(Ptac::mucA'B) were mated
with GY306 (HfrH). Prior to mating, expression of MucA'B in the
recipients was induced with 0 or 500 µM IPTG. Open bars,
recipients without MucA'B; gray and dark bars, recipients
expressing MucA'B from a Ptac promoter at
basal or IPTG-induced levels, respectively.
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These data suggest that MucA'B and UmuD'C may act
synergistically in inhibiting recombination. MucAB and UmuDC have
diverged during evolution and do not form mixed complexes
(29, 33). The observed synergism of MucA'B and UmuD'C
can be accounted for if each complex contacts a RecA polymer at
distinct sites. Indeed, if each of the two mutagenic complexes
binds independently to a RecA polymer at distinct sites, one can
explain why when the interaction sites of one complex are saturated,
the other complex can still bind to a RecA polymer, inhibiting further recombination.
RecA[UmuR] proteins are sensitive to recombination inhibition
by MucA'B.
We asked whether the recently isolated mutant
RecA[UmuR] proteins, which overcome UmuD'C recombination
inhibition (38), are also sensitive to recombination
inhibition by MucA'B. We tested mutants RecA D112G, S117F, V247L,
and S44L, which have mutations located at the three sites where the
[UmuR] amino acid changes have been shown to occur: the tail domain
(D112G and S117F), the head domain (V247L), and the core of a RecA
monomer (S44L) (38). RecA S117F, which is dominant over
RecA+ in conferring Umu resistance (38), was
used in a diploid combination with RecA+.
The sensitivity of the recA[UmuR] bacteria to MucA'B
was assessed by measuring the efficiency of conjugational
recombination in derivatives expressing the MucA'B complex
from a Ptac promoter either at a low basal level
or at an elevated IPTG-induced concentration (Fig.
5). As seen in Fig. 5, the
RecA[UmuR] proteins were sensitive to MucA'B recombination
inhibition. RecA mutants D112G, S117F (in the diploid combination with
RecA+), and V247L behaved like wild-type RecA+.
RecA S44L partially escaped recombination inhibition, yet recombination was still decreased 10-fold at an elevated MucA'B concentration (Fig. 5).
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FIG. 5.
RecA[UmuR] proteins are sensitive to recombination
inhibition by MucA'B. Derivatives of GY9054 (F
leuB6 recA umuDC/mini-F-recA+,
-recA410, -recA411, or -recA415) and
of GY9743 (F leuB6
recA+
umuDC/mini-F-recA1730) carrying plasmid GY9411
(Ptac::mucA'B) (gray and dark
bars) or the vector pTrc99A (open bars) were crossed with GY10229
(HfrH). Prior to mating, expression of MucA'B in the
recipients was induced with 0 (gray bars) or 500 (dark bars) µM
IPTG.
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The MucA'B complex promotes efficient mutagenesis in
recA[UmuR] bacteria.
It has been shown that the
recA[UmuR] bacteria have a reduced UV mutability, a defect
that has been attributed to a poor interaction of a RecA[UmuR]
polymer with the UmuD'C complex (38). If the MucA'B
complex interacts rather efficiently with a RecA[UmuR] protein,
one might expect that it would alleviate the mutability defect of
a recA[UmuR] strain. Indeed, recA[UmuR]
bacteria carrying either pKM101, the natural mucAB
plasmid, or plasmid pRW294, which expresses the MucA'B
complex at a low level, promoted UV mutagenesis efficiently (Fig.
6). Mutant recA S44L,
D112G, and V247L displayed the same level of Muc-promoted
mutability as recA+. The
recA+/recA S117F heterogenote, which is
the most affected in Umu-promoted mutagenesis (38), also
recovered about 60% of the wild-type mutability in the presence of
MucA'B.
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FIG. 6.
Efficient Muc-promoted mutagenesis in
recA[UmuR] bacteria. Derivatives of GY9054
(recA umuDC/mini-F-recA+,
-recA410, -recA411, or -recA415) and
of GY9743 (recA+
umuDC/mini-F-recA1730) carrying plasmid pKM101
(left panel) or plasmid pRW294 (right panel) were assayed for
Arg+ reversion after exposure to 20 J of UV light
m2.
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DISCUSSION |
Antirecombination activity of mutagenic complexes.
We demonstrated here that the MucA'B complex from
plasmid pKM101 inhibits homologous recombination as its cellular
homolog UmuD'C does. An antirecombination activity might be a general property shared by other members of the family of Umu-like mutagenesis proteins and might be related to their common ability to interact with
a RecA polymer (7, 13, 23).
Recombination and mutagenesis are two repair processes that take place
sequentially on the single-stranded discontinuities (DNA gaps) arising
after replication of damaged DNA (30). The two processes
require single-stranded DNA around which a RecA polymer wraps. Most
RecA-coated gaps are first mended by recombinational repair. At the
remaining gaps, the binding of a mutagenic complex to RecA prevents
recombination from occurring and switches repair into a mutagenic
pathway. The induced mutability might be the product of the capacity of
a mutagenic complex to assist DNA polymerase in trans-lesion
synthesis coupled with its antagonistic action on recombination.
The enhanced mutagenic activity of the MucA'B complex is not
correlated with an enhanced antirecombination activity.
The
MucAB proteins are more proficient in mutagenesis than the related
UmuDC proteins (5, 23). This difference has been attributed
to more efficient processing of MucA than of UmuD (17). Our data demonstrate that the MucA'B complex possesses an inherent superior capacity to stimulate cellular mutagenesis.
Two sets of interactions might be specifically relevant to the
MucA'B mutagenic activity: (i) an interaction of MucA'B with a
RecA polymer (13), which might target the MucA'B complex
at a DNA lesion while inhibiting recombination, and (ii) an interaction of MucA'B with subunits of DNA polymerase III and with
single-stranded-DNA-binding protein (31, 42), which might
account for the function of the MucA'B complex to stimulate
trans-lesion synthesis. A comparison of our present data
with those of Sommer et al. (37) and of Boudsocq et al.
(6) indicates that quantitatively MucA'B does not
inhibit recombination more than UmuD'C does. Thus, it is unlikely that
the MucA'B complex binds a RecA polymer with a greater affinity. The enhanced mutagenic potential of the MucA'B complex seems to be
more correlated with an increased efficiency in helping DNA polymerase
to synthesize across a large range of DNA lesions. Such a view is
supported by the work of Lawrence et al. (24), who showed
that the MucA'B complex per se is more active than UmuD'C in
promoting replication past a defined abasic site carried by a
single-stranded DNA vector.
MucA'B and UmuD'C may interact with a RecA polymer at different
sites.
The MucA'B complex also relies on the RecA chaperoning
function to be active in mutagenesis (reference 36
and unpublished results). It is thought that, like UmuD'C, the
MucA'B complex is targeted at a DNA lesion through its interaction
with a RecA polymer (3, 13). Our data suggest that the two
mutagenic complexes might contact a RecA polymer at distinct sites.
We observed, indeed, that the recently isolated RecA[UmuR]
proteins, which are resistant to UmuD'C inhibition of
recombination (38), are sensitive to recombinational
inhibition by MucA'B and support MucA'B-promoted
mutagenesis efficiently. The mutant RecA[UmuR] proteins seem to
interact efficiently with MucA'B. An interaction of MucA'
with a RecA[UmuR] polymer, namely, RecA S117F, has been
demonstrated in vitro (13).
Two hypotheses may account for the interaction of MucA'B with a
mutant RecA[UmuR] polymer: (i) MucA'B proteins may have
evolved to be functional in bacteria divergent from Escherichia
coli and may bind RecA with a relaxed specificity, and (ii) the
binding of MucA'B to RecA may involve specific interactions,
with MucA'B and UmuD'C contacting a RecA polymer at distinct sites.
Our findings that MucA'B and UmuD'C act synergistically in
inhibiting recombination support the second hypothesis. If each of the
two mutagenic complexes binds a RecA polymer independently at distinct
sites, then one can explain why when the interaction sites of one
complex are saturated, the other complex can still bind to a RecA
polymer, inhibiting further recombination.
A test of these models awaits the isolation of recA
mutations specifically altering RecA-MucA'B interactions. It is
interesting that RecA S44L was less sensitive to recombination
inhibition by MucA'B than the other RecA[UmuR] proteins. The
S44 amino acid lies in the core of a RecA monomer (39).
Should the S44L amino acid change have a far-reaching effect on the
overall structure of a RecA monomer, it might partially alter a
putative MucA'B binding site.
Elevated levels of Muc proteins are expressed from the plasmid
pKM101.
In the course of this work, we found that the
concentrations of the Muc proteins expressed from the natural
plasmid pKM101 in an SOS-induced cell are particularly elevated, taking
into account the low-level production of their chromosomally encoded Umu counterparts. For instance, the level of MucA expressed
from pKM101 in lexA(Def) bacteria, devoid of LexA, was
18 times higher than that of UmuD (45,000 MucA molecules per
cell versus 2,500 UmuD molecules per cell) (Table 3) (45).
We speculate that an elevated level of the plasmid-encoded mutagenic
proteins might be required for an additional plasmid function. pKM101
belongs to the IncN group of broad-host-range conjugative
plasmids. We suggest that the muc genes may become transiently induced during conjugation and may facilitate the installation of pKM101 into a variety of host cells. Although they are
not essential for conjugal transfer under laboratory conditions, the
Muc proteins may enhance the fitness of plasmid transmission if
they speed up replication of the transferred plasmid single-stranded
DNA by interacting with the host DNA polymerase. Interestingly, in
pKM101 the muc operon is located in the leading region,
the first segment that enters the recipient cell during conjugation
(4). The leading regions of many conjugative plasmids carry
installation genes that are thought to promote the establishment of an
immigrant plasmid into a new host cell (2, 14, 20).
Bacterial conjugation mediates genetic exchange not only between cells
of the same species but also between members of distantly related and
even unrelated genera. This promiscuous gene transfer provides a
mechanism for making available a huge pool of genes for bacterial
evolution (for a review, see reference 15). In this
regard, it is significant that half of the mutagenic repair genes
are carried by conjugative plasmids (40).
We benefited from the qualified assistance of D. Bouillon and G. Coste in genetic and molecular biology experiments. We thank M. Pierre
for her help in preparing the manuscript. We are grateful to R. Woodgate for providing anti-MucA and -A' and anti-MucB
antibodies as well as valuable strains and to Z. Livneh for the
generous gift of purified MucA, MucA', and MucB proteins.
We thank R. Woodgate, A. Roca, and P. Stragier for helpful discussions
and comments and R. Devoret for his constant advice.
C. Venderbure is the recipient of a fellowship from the Ministère
de l'Education Nationale de la Recherche et de la Technologie. F. Boudsocq benefited from fellowships from the Association pour la
Recherche sur le Cancer and from the Fondation pour la Recherche Médicale. This work was supported by grants to A. Bailone from the Ligue Nationale contre le Cancer, from the Association pour la
Recherche sur le Cancer (no. 9601), and from the Ministère de
l'Education Nationale de la Recherche et de la Technologie (AS
Radiobiologie 98-15) and by funds to UMR 216 from the Institut Curie
and from the Centre National de la Recherche Scientifique.
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Abstract
Introduction
Present address: NIH, Bethesda, MD 20892-2725.
