From the Department of Microbiology and Immunology,
Feinberg School of Medicine, Northwestern University, Chicago,
Illinois 60611 and the ¶ Division of Biological Sciences, Section
of Microbiology and of Molecular and Cellular Biology, Center for
Genetics and Development, University of California, Davis, California
95616
Received for publication, October 14, 2002
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ABSTRACT |
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In Escherichia coli the RecA protein
plays a pivotal role in homologous recombination, DNA repair, and SOS
repair and mutagenesis. A gene designated recX (or
oraA) is present directly downstream of recA in
E. coli; however, the function of RecX is unknown. In this
work we demonstrated interaction of RecX and RecA in a yeast two-hybrid
assay. In vitro, substoichiometric amounts of RecX strongly
inhibited both RecA-mediated DNA strand exchange and RecA ATPase
activity. In vivo, we showed that recX is under control of the LexA repressor and is up-regulated in response to DNA
damage. A loss-of-function mutation in recX resulted in decreased resistance to UV irradiation; however, overexpression of RecX
in trans resulted in a greater decrease in UV resistance. Overexpression of RecX inhibited induction of two din
(damage-inducible) genes and cleavage of the
UmuD and LexA repressor proteins; however, recX
inactivation had no effect on any of these processes. Cells overexpressing RecX showed decreased levels of P1 transduction, whereas
recX mutation had no effect on P1 transduction frequency. Our combined in vitro and in vivo data indicate
that RecX can inhibit both RecA recombinase and coprotease activities.
The Escherichia coli RecA protein plays a central role
in homologous recombination and is required for induction of the SOS pathway of DNA repair and mutagenesis. A fundamental step in both homologous recombination and SOS response induction is the formation of
a RecA-ssDNA1-ATP
nucleoprotein filament. In this form, RecA protein can act as a
recombinase by mediating pairing and promoting strand exchange between
single-stranded DNA and another homologous DNA molecule, a process
important for phage transduction, conjugation, and DNA repair (1, 2). RecA also functions as a coprotease to activate the SOS
response (3). The LexA protein represses transcription of over 30 SOS
genes in the E. coli SOS regulon (4). DNA damage is believed
to produce regions of ssDNA upon which RecA forms a nucleoprotein
filament. RecA coprotease activity facilitates self-cleavage of
the LexA repressor protein, enhancing expression of SOS genes. RecA
coprotease activity is also responsible for cleavage of the UmuD
protein, which is involved in SOS mutagenesis. It is assumed that the
repair of damaged DNA gradually removes the signal (ssDNA) needed for
nucleoprotein filament formation, resulting in the re-accumulation of
LexA pools and subsequent repression of the SOS genes, thereby
resetting the SOS system (3). In contrast to this essentially passive
process, overexpression of the E. coli DinI protein has been
shown to inhibit induction of the SOS response through inhibition of
RecA coprotease activity (5), and a dinI knockout showed
both increased UmuD cleavage and SOS mutagenesis, suggesting that DinI
may play an active role in turning off or modulating the SOS response
(5, 6).
An open reading frame, originally designated oraA, is
located directly downstream of recA and upstream of
alaS in E. coli (7) and shows sequence similarity
to RecX proteins from Gram-positive and Gram-negative bacteria (8).
recX genes are located downstream of recA or
overlapping recA (8-12) or occasionally elsewhere in the
chromosome, as in Neisseria gonorrhoeae and Bacillus
subtilis (13). Overexpression of homologous RecA proteins from
plasmid constructs is deleterious in the absence of recX in
Pseudomonas aeruginosa, Streptomyces lividans,
Mycobacterium smegmatis, and Xanthomonas oryzae
(9, 11, 14, 15), suggesting a role for RecX in down-regulating RecA
expression or activity. Consistent with these observations,
Mycobacterium tuberculosis RecX was recently shown to
inhibit RecA-promoted DNA strand exchange and ATP hydrolysis in
vitro (16). Although a recX mutation in S. lividans did not affect homologous recombination or transcription
of recA (11), mutation of recX in X. oryzae resulted in decreased RecA levels (15). In contrast, a
N. gonorrhoeae recX mutant exhibited deficiencies in all
RecA-mediated processes but did not affect RecA levels, leading to the
conclusion that RecX enhances RecA activity in N. gonorrhoeae (13). This collection of phenotypes led us to investigate the role of E. coli RecX in vitro and
in vivo. The data presented here demonstrate that E. coli RecX can strongly inhibit both RecA recombinase and
coprotease activities in vitro and in vivo.
Strains, Plasmids, Media, and Chemicals--
New strains were
constructed using P1 transduction (17) or as described. Strains and
mutant alleles used were DH5 DNA Manipulations and Analysis--
Standard procedures were
performed as described previously (26). Extraction of DNA from bacteria
and agarose gels was performed using Qiagen kits. Enzymes were used
according to manufacturers' directions (Promega, New England BioLabs).
Sequencing reactions were performed using the Big-Dye Terminator cycle
sequencing kit (PerkinElmer Life Sciences), and sequencing products
were separated on an ABI model 377 automated DNA sequencer.
Yeast Two-hybrid Analysis--
Yeast two-hybrid analysis was
performed using the Matchmaker GAL4 two-hybrid system 3 as described
(Clontech). E. coli recA was
amplified from plasmid pERA by PCR using Pfu polymerase
(Stratagene) with primers ECRECAFOR
5'-TTACATATGGCTATCGACGAAAACAAACAG-3', which introduces an NdeI site (underlined), and ECRECAREV
5'-GACTTAAAAATCTTCGTTAGTTTCTGC-3'. E. coli
recX was similarly amplified using primers ECRECXFOR
5'-TTACCATGGACATGACAGAATCAACATCCC-3', which
introduces a NcoI site (underlined), and ECRECXREV
5'-TTATCAGTCGGCAAAATTTCGCC-3'. The gel-purified PCR fragments
were cloned into pCR-Blunt (Invitrogen) and sequenced. The genes were
further subcloned into NdeI/BamHI-digested (for
recA) and NcoI-EcoRI-digested (for
recX) vectors pGADT7 and pGBKT7
(Clontech), yielding constructs pGADT7-RecX,
pGADT7-RecA, pGBKT7-RecX, and pGBKT7-RecA. Clones were sequenced to
verify maintenance of proper reading frame, and protein expression of all relevant constructs in yeast was demonstrated by Western blot analysis (data not shown).
Construction of Plasmids pET/HisRecX,
pGCC4/recX, and pGCC4/HisRecX--
Plasmid
construct pET/HisRecX was used to overexpress RecX as an N-terminal
His-tagged protein. The recX coding region was amplified by
PCR from plasmid pERA2 using
primers ECRXFORNHE, which introduces a NheI site
(underlined) (5'-GTAGCTAGCATGACAGAATCAACATCC-3'), and
ECRX1R (5'-GCTGGTAACTGAAAAGTGGG-3') with Pfu polymerase. The
gel-purified PCR product was ligated to pCR-Blunt (Invitrogen) to yield
pCRHisRecX, and the resulting clone was sequenced to verify that no
mutations had been introduced. The recX insert was isolated
by NheI-HindIII digestion and ligated to
NheI-HindIII-digested pET28a vector (Novagen) to
yield construct pET/HisRecX.
The E. coli recX gene was cloned under control of
the lac promoter in plasmid pGCC4, a high copy number
plasmid with a ColE1 origin of replication (27). recX was
amplified from plasmid pERA by PCR using primers RECXFORPAC, which
introduces a PacI site (underlined)
(5'-GTAGGTTAATTAAGTTGTAAGGATATGCCA-3'), and RECXREV
(5'-AGTCGCTAGCAATACCGTATGCGTTCAGTCG-3') using Pfu polymerase (Stratagene). The fragment was digested with PacI,
gel-purified, ligated to PacI-PmeI-digested
pGCC4, and the resulting clone was sequenced. Plasmid pGCC4/HisRecX was
created by ligating the blunt-ended XbaI/HindIII
fragment of pET/HisRecX to PmeI-digested pGCC4. Expression of similar levels of His-tagged RecX (HisRecX) and RecX proteins from
vector pGCC4 in strain AB1157 Protein Purification--
HisRecX protein was
overexpressed in E. coli BL21(DE3) (pET/HisRecX) cells.
Cultures (500 ml) were grown at 37 °C to mid-exponential phase (A600 ~ 0.4), IPTG (1 mM) was added to induce expression of HisRecX, and the
culture was grown an additional 1.5 h. Cells were harvested by
centrifugation (10,000 × g, 10 min) and resuspended in
1/10 volume column binding buffer (5 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl (pH 7.9)) amended with
0.1% Triton X-100. Cells were disrupted by sonication on ice for three
1-min pulses, with 1-min cooling between pulses, with a Vibra-Cell
probe sonicator at output setting 4, 50% duty cycle (Sonics & Materials, Inc.). Soluble proteins were separated from cell debris and
insoluble proteins by centrifugation (14,000 × g, 20 min) and by subsequent passage through a 0.45-µm filter. Clarified
supernatant was applied to a HisBind Quick column (Novagen) that had
been equilibrated with 15 ml of column binding buffer, and the column
was washed with 50 ml of column binding buffer and 25 ml of column wash
buffer (60 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl (pH 7.9)). HisRecX protein was eluted from the
column with 100 mM EDTA, 0.5 M NaCl, 20 mM Tris-HCl (pH 7.9), and fractions containing HisRecX were identified via SDS-PAGE. HisRecX-containing fractions were combined and
dialyzed against three changes of a 200-fold excess of 20 mM Tris-HCl (pH 7.9) over 24 h at 4 °C and
subsequently concentrated in a Centriprep-40 concentrator (Millipore).
Protein concentration was determined by BCA assay (Pierce), and protein
purity was assessed to be >98% by quantitative scanning of Coomassie
Blue-stained SDS-PAGE gels. For protein used in activity assays,
glycerol was added to 20% final concentration and protein was
flash-frozen on dry ice/EtOH. RecA and SSB proteins were prepared as
described previously (28).
DNA Strand Exchange Assay--
The agarose gel assay for DNA
strand exchange was conducted and visualized as described (29).
Reactions contained 25 mM Tris acetate (pH 7.5), 10 mM magnesium acetate, 0.1 mM dithiothreitol, 2 mM phosphoenolpyruvate, 10 units of pyruvate kinase per
milliliter, 7 µM M13 ssDNA, 1 mM ATP, 0.64 µM SSB, 4.2 µM RecA, and 10 µM M13 replicative form dsDNA linearized with
EcoRI, and varying concentrations of RecX.
ATP Hydrolysis Assay--
The ssDNA-dependent
hydrolysis of ATP by RecA was measured as described using a coupled
enzymatic assay (30). Reactions contained 7 µM M13 ssDNA,
4.2 µM RecA, 0.64 µM SSB (where indicated), and varying amounts of RecX. RecA protein was added to M13 circular ssDNA either before RecX or following 10-min incubation of RecX with
M13 circular ssDNA. When SSB was included, it was added 1 min after the
addition of RecA.
Insertional Inactivation of recX--
We created a 368-bp
non-polar internal deletion of recX using the method
described by Datsenko and Wanner (25). Gene deletions using this system
are engineered to introduce stop codons in all six reading frames and
an idealized ribosome binding site and start codon for downstream gene
expression at the site of gene deletion. The gene disruptions created
using this system have been shown to be non-polar (25). A PCR product
was generated from plasmid pKD4 (25) using primers H15
(5'-AATCAACATCCCGTCGCCCGGCATATGCTCGCCTGTTGGATCGTGTGTAGGCTGGAGCTGCTTC-3') and H23
(5'-GCAAAATTTCGCCAAATCTCCTGGATATCTTCCATCAGATAGCCCATATGAATATCCTCCTTAG-3'). This PCR product contains a kan marker flanked by short
regions of homology to the recX gene at the 5'- and 3'-ends
(underlined in primer sequences). We transformed strain AB1157 (pKD46)
with this PCR product and selected for KanR colonies to
identify insertions into recX, yielding strain
AB1157 UV Resistance Assays--
Overnight cultures were diluted into
LB or LB-Erm (1.5 mM IPTG) and grown to early stationary
phase (A600 = 1.2), serially diluted, and
spot-plated onto the appropriate LB agar. Plates were exposed to 0, 2.5, 5.0, 7.5, and 10 J/m2 UV light (UV Stratalinker 1800, Stratagene).
P1 Phage Transduction--
Overnight cultures were diluted into
LB or LB-Erm containing 5.0 mM CaCl2 and grown
until late log phase (A600 = 0.8). IPTG (1.5 mM) was added to cultures containing plasmids, and cultures were grown for an additional 30 min. P1 transduction was performed as
described (17) using phage grown on Hfr Cavalli.
LexA and UmuD Cleavage Assays--
For LexA cleavage
assays, overnight cultures were diluted into LB or LB-Erm (1.5 mM IPTG) and grown to A600 = 0.4, and cultures were supplemented with chloramphenicol (100 µg/ml) and
incubated for an additional 10 min. For cultures containing plasmids
pGCC4 and pGCC4/recX, aliquots were taken
(t0), the remainder of the culture was exposed
to 8 J/m2 UV irradiation, and additional aliquots were
taken from the irradiated samples after 5, 15, 30, 60, and 90 min. For
strains AB1157 and AB1157
For UmuD cleavage assays, overnight cultures were diluted into LB-Spc
or LB-Erm-Spc (1.5 mM IPTG). For this analysis, all strains
additionally contained plasmid pRW362 (umuD) to facilitate UmuD detection. Cells were grown to A600 = 0.4, a t0 aliquot was taken, and MMC was added (0.2 µg/ml). Aliquots were taken after 80, 120, 160, 200, and 240 min.
Protein concentration of samples was determined by BCA assay (Pierce).
Western Analysis--
Western analysis and protein
quantification was performed as described (13), except for exceptions
noted. Samples were run on 15% SDS-PAGE gels (for RecX and LexA), 17%
(for UmuD), or 12% (for RecA and all constructs in yeast), and
subsequently developed using ECL or ECL Plus Western blotting protocols
(Amersham Biosciences). Antibodies against HisRecX, raised in a rabbit
using the Polyquik method (Zymed Laboratories), were used at a 1:600
dilution. Anti-LexA antibodies (Invitrogen), anti-UmuD antibodies
(provided by R. Woodgate, National Institutes of Health, Bethesda, MD),
anti-RecA antibodies (provided by M. Cox, University of
Wisconsin-Madison), anti-c-myc and anti-HA antibodies (Roche Molecular
Biochemicals) were used at 1:5,000 dilutions. Secondary goat
anti-rabbit IgG, goat anti-mouse IgG, and rabbit anti-rat IgG
antibodies conjugated to horseradish peroxidase were used at 1:5,000 to
1:15,000 dilutions (Roche Molecular Biochemicals). Membranes were
either exposed to Kodak film or detected using a ChemiImager (Alpha
Innotech Corp.). Subsequent densitometric analyses were performed using ImageQuaNT (Amersham Biosciences) or Alpha Ease (Alpha Innotech) software, respectively.
RecX and RecA Physically Interact--
In several bacterial
systems, overexpression of RecA proteins from plasmid constructs is
deleterious in the absence of recX (9, 11, 14, 15),
suggesting that RecX may down-regulate RecA activity or expression,
possibly by interacting with the RecA protein. To investigate the
direct association of E. coli RecX and RecA proteins, a
yeast two-hybrid analysis was performed. Both recA and
recX genes were cloned into vectors pGBKT7 (TRP1 marker) and pGADT7 (LEU2 marker), yielding constructs
pGBKT7-RecA, pGBKT7-RecX, pGADT7-RecA, and pGADT7-RecX that express
fusion proteins with Gal4 DNA binding or activation domains (described under "Experimental Procedures"). The yeast two-hybrid reporter strain AH109 contains ADE2, HIS3, and
MEL1 reporter genes that are expressed only when a
functional Gal4 protein is reconstituted by an interaction between the
activation domain and the DNA binding domain fusion proteins. AH109
cells carrying plasmid pairs pGBKT7-RecA/pGADT7-RecX or
pGBKT7-RecX/pGADT7-RecA grew robustly on media lacking histidine, tryptophan, and leucine, and showed a blue color on media supplemented with X- RecX Inhibits RecA Protein-promoted DNA Strand Exchange
in Vitro--
To investigate the functional significance of RecX
interaction with RecA, we measured the effect of RecX on RecA
recombinase activity in vitro. RecA-promoted DNA strand
exchange was measured in the presence or absence of RecX. RecX
completely abolished formation of joint molecules over a 60-min time
course between homologous circular ssDNA and linear dsDNA molecules at
a RecX:RecA molar ratio of 1:2.2 (Fig.
2). In the absence of RecA, RecX neither catalyzed the formation of joint molecules nor degraded DNA (data not
shown), demonstrating that the effect of RecX on RecA activity is not
simply due to degradation of DNA substrates. Titrating the level of
RecX protein in the strand exchange reaction, complete inhibition of
joint molecule formation was observed at a RecX:RecA molar ratio of
1:44, minimal joint molecule formation was seen at a 1:88 ratio, and
resolution into a nicked circular form was never observed, even at a
RecX:RecA molar ratio of 1:707 (Fig. 2). These data suggest that
sub-stoichiometric levels of RecX inhibit both the initial pairing of
homologous molecules, and, to a greater degree, subsequent branch
migration.
RecX Inhibits RecA ssDNA-dependent ATPase
Activity--
Formation of a RecA-ssDNA-ATP nucleoprotein filament is
accompanied by the subsequent hydrolysis of ATP by RecA (ATPase
activity) and reflects the amount of active nucleoprotein filament
formed. We therefore assessed the effect of RecX on RecA ATPase
activity in the presence or absence of SSB, preincubating RecX with
ssDNA before the addition of RecA. In the absence of SSB, RecX
decreased ATPase activity 20% when present at a 1:70 RecX:RecA molar
ratio (60 nM RecX), 50% when present at a 1:14 RecX:RecA
molar ratio (300 nM RecX), and 85% when present at a 1:8
molar ratio (500 nM RecX) (Fig.
3). In the presence of SSB, the effects
of RecX were more dramatic, with ATPase activity nearly completely
abolished (decreased 98%) by a RecX:RecA molar ratio of 1:70,
approximately the level where minimal joint molecule formation was seen
in the in vitro strand exchange reaction. We observed the
same effect of RecX on ATPase activity when RecX was added to the
reaction after preincubation of RecA and ssDNA (data not shown).
Therefore, the inhibitory effect of RecX is enhanced in the presence of
SSB but is independent of the time of RecX addition to the
reaction.
RecX Is Part of the SOS Regulon--
The strong inhibitory effect
of RecX on RecA activity in vitro suggested that RecX could
have dramatic effects modulating RecA activities in vivo as
well. Therefore, we began to characterize the E. coli
recX gene to determine its role in the bacterial cell. In
E. coli, recX is directly preceded by neither a
canonical promoter sequence nor an SOS box, but it is located 76 bp
downstream of the SOS-regulated recA gene, suggesting that
recX could be co-transcribed with recA and,
therefore, under control of LexA. Western blot analysis revealed
expression of a ~19-kDa band barely detectable in strain AB1157 that
increased in intensity after treatment with mitomycin C (MMC) (Fig.
4) or exposure to UV light (data not
shown). This band was undetectable in the MMC-treated
RecX Alters UV Resistance--
Because recX was induced
upon DNA damage, suggesting a possible role in DNA repair, we tested
the effect of the
The amount of RecX protein present in various bacterial cells was
determined in a semi-quantitative immunoblotting analysis. Serial
dilutions of purified RecX protein were used as a standard for
comparison against cell extracts made from a known quantity of
bacterial cells. We estimated the basal number of RecX molecules in
AB1157 to an average of <50 molecules per cell. After SOS induction of
AB1157 with MMC (2 h), these averaged 800 molecules of RecX per cell.
Cells carrying pGCC4/recX (induced with IPTG) averaged 105 RecX molecules per cell, whereas cells carrying
pGCC4/recX (uninduced with IPTG) averaged 103
RecX molecules (data not shown). These results indicate that both
overexpression and loss of RecX decrease UV resistance.
RecX Overexpression Inhibits Induction of the SOS Response--
To
determine whether RecX overexpression alters UV resistance by
inhibiting induction of the SOS response, bacterial strains that
contain SOS-regulated chromosomal fusions of either
sulA::lacZ or
dinD1::lacZ were transformed with
either pGCC4 or pGCC4/recX, and gene expression was measured
by quantifying
The observed inhibition of SOS induction by RecX
overexpression could be due to effects on either RecA activity or RecA
levels. Although our in vitro experiments suggested that
RecX affects RecA activity, we wanted to additionally test this
in vivo. Western blot analyses showed no difference in basal
RecA protein levels between strains AB1157 and AB1157 RecX Overexpression Inhibits LexA and UmuD Cleavage--
Because
RecX has only minor effects on basal RecA protein levels, we tested the
hypothesis that RecX overexpression inhibits SOS induction by
inhibiting RecA coprotease activity. SOS induction requires activated
RecA to function as a coprotease to facilitate LexA self-cleavage,
thereby derepressing genes of the SOS regulon. RecA coprotease activity
is also responsible for the cleavage that converts UmuD to UmuD', the
activated form of the protein that is involved in SOS-induced
mutagenesis. We used Western blot analysis to monitor degradation of
the LexA repressor protein after exposure to UV light under conditions
where de novo protein synthesis was inhibited by
chloramphenicol. In cells carrying pGCC4/recX, LexA was
detected 90 min after UV exposure (Fig.
7A), whereas LexA was
completely degraded in cells carrying the pGCC4 vector after 30 min. In
contrast, there were no differences in the extent or rate of cleavage
of LexA in strains AB1157 and AB1157
The effect of recX on UmuD cleavage was measured using
derivatives of strain DE192 (see "Experimental Procedures"), which carries a lexA51 (Def) mutation resulting in constitutive
expression of LexA-regulated genes. In these strains, DNA damage
triggers cleavage of UmuD. We observed decreased cleavage of UmuD after 200-min treatment with MMC in cells carrying pGCC4/recX
relative to cells carrying the vector alone (Fig. 7B). No
differences in UmuD cleavage were observed between strains DE192 and
DE192 RecX Overexpression Inhibits P1 Transduction--
Because we had
observed inhibition of RecA-mediated DNA strand exchange by
substoichiometric quantities of RecX in vitro, we wanted to
test the effect of RecX on RecA recombinase activity in
vivo. The effect of RecX on RecA-mediated homologous recombination was quantified in vivo with P1 transduction assays. The P1
transduction frequency of proline and leucine prototrophic markers was
the same in strains AB1157 and AB1157 The results presented in this study show that E. coli
RecX can modulate RecA activities through direct physical interaction with RecA. In vitro studies established that RecX inhibits
RecA recombinase and ATPase activities at substoichiometric levels and
suggest mechanistic bases for this inhibition. In vivo
studies where RecX was overexpressed corroborated these results,
showing that RecX overexpression strongly inhibits RecA recombinase as well as RecA coprotease activities.
The inhibition of RecA ATPase and recombinase activities in
vitro by substoichiometric amounts of RecX protein, coupled with the interaction of RecX and RecA in a yeast two-hybrid assay, suggest
several mechanisms for RecX inhibition of RecA activities in
vitro. Both RecA coprotease and recombinase activities require the
formation of a RecA-ssDNA-ATP nucleoprotein filament. Therefore, affecting either the formation or integrity of the nucleoprotein filament will affect both activities of RecA. In gel-shift assays, we
observed RecX binding to 100-mer ssDNA and dsDNA only at RecX concentrations of >1 µM (data not shown), a level too
high to account for the observed inhibition of strand exchange.
Therefore, we do not favor the models that RecX binds to ssDNA, thereby
disrupting or impeding formation of the nucleoprotein filament, or that
RecX binds to dsDNA, blocking homologous DNA exchange. During
homologous recombination, dsDNA is believed to lie within the deep
helical groove of the RecA nucleoprotein filament (32). The LexA
repressor protein may also bind within this groove (33) and, possibly, the UmuD2'C complex (34) and UmuD protein (35). Therefore, a second model is that RecX interacts preferentially with RecA within
the deep helical groove, thereby blocking access to or displacing the
above substrates, as is believed to occur with the DinI protein (6).
Although this model suggests how RecX could inhibit both RecA
coprotease and recombinase activities through direct interaction with
RecA, it accounts for neither the observed inhibition of RecA ATPase
activity by RecX nor the effect of SSB on the reaction. Thus, a favored
model is that RecX binds to RecA and diminishes the ability of RecA to
bind ssDNA or ATP. The interaction of RecA and RecX may occur with
either free RecA protein or RecA within the nucleoprotein filament. The nearly complete inhibition (>98%) of RecA ATPase activity by RecX in
the presence of SSB, compared with the less dramatic inhibition (~20%) in the absence of SSB, suggests a role for SSB in the
interaction of RecA and RecX as well. Similar to what was proposed to
occur with the uncleavable LexA repressor protein (36), the interaction of RecX and RecA may disrupt the equilibrium of RecA and SSB binding to
ssDNA, favoring the binding of SSB to ssDNA and resulting in collapse
of the nucleoprotein filament and inhibition of ATPase activity. The
observed effect of RecX on RecA recombinase activity in
vitro could be due to the subsequent creation of gaps in the RecA-ssDNA-ATP nucleoprotein filament, blocking branch migration in
particular, where a RecX:RecA molar ratio of 1:707 inhibited resolution
of intermediates into a nicked-circular form (Fig. 2) but having a much
smaller effect on nascent joint molecule formation, where a RecX:RecA
molar ratio of 1:44 was required for inhibition (Fig. 2). RecX-mediated
collapse of the nucleoprotein filament can also explain the decreased
cleavage of LexA and UmuD proteins and the decreased P1 transduction
frequency observed in vivo during RecX overexpression. The
profound decrease in UV resistance when RecX was overexpressed could be
due to a combination of suppression of SOS induction, through
inhibition of RecA coprotease activity, and inhibition of
recombinational DNA repair, both a result of nucleoprotein filament collapse.
E. coli RecX is a potent inhibitor of RecA activity in
vitro when supplied at substoichiometric levels and in
vivo when overexpressed, but the only phenotype observed in the
An alternate role for RecX is suggested by studies in other bacteria.
An recX mutant of N. gonorrhoeae was decreased in
the RecA-mediated processes of DNA repair, pilus antigenic variation, and DNA transformation, suggesting that RecX may enhance RecA activity
in this organism (13). Interestingly, N. gonorrhoeae is one
of the few bacteria where recX is not found near
recA (13), and N. gonorrhoeae lacks an SOS
response (41). In X. oryzae, where recX is
located downstream of recA, a recX mutant showed a 50% decrease in RecA levels relative to the parent strain (15). Finally, investigations of the E. coli RecX and RecA
proteins heterologously expressed in N. gonorrhoeae
suggested that RecX (or recX) may either stabilize the RecA
protein or recA transcript when present at low levels (19).
Therefore, RecX may either have multiple activities in a particular
bacterial species, or RecX may show variable activity between species.
The RecX protein from M. tuberculosis (MtRecX)
was recently characterized in vitro and was found to have
profound inhibitory effects on RecA activity at substoichiometric
levels and to interact with RecA (16). The MtRecX was also able to
inhibit EcRecA activity in vitro, although not as
efficiently as it did MtRecA activity (16). In combination with our
current study, this work suggests that the M. tuberculosis
and E. coli RecX proteins are homologues. However, the
relative activity of MtRecX may differ slightly from that of E. coli RecX (EcRecX). Whereas we observed complete inhibition (>98% decrease) of ATPase activity at an EcRecX:EcRecA molar ratio of
1:70, ATPase activity was decreased 86% at a MtRecX:MtRecA molar ratio
of 1:1.6 (16). The authors speculate that the biological role of MtRecX
is to quell inappropriate recombinational repair during normal DNA
metabolism; however, a recX mutant has not been generated in
M. tuberculosis, so the function of MtRecX in
vivo remains untested (16).
Although the exact biological role of RecX in any bacterium
remains unclear, the importance of RecX for RecA activity is
underscored by its conserved location with recA in myriad
bacterial genomes (8-12, 15). Because E. coli RecX has
potent inhibitory effects on RecA activities both in vitro
and in vivo, and because RecX is up-regulated during the SOS
response in many bacteria (11, 12, 15, 31, 39), this suggests that RecX
has some regulatory role during the SOS response, which has yet to be
elucidated. The identification of other proteins that modulate RecA
activities emphasizes the biological importance of regulating both the
SOS response and homologous recombination. psiB, which is
found on many conjugative plasmids near oriT, the origin of
conjugative transfer, possesses anti-SOS functions and anti-recombinase
activities (42, 43). The location of psiB hints at its
hypothesized function: to prevent ssDNA that is transferred upon
conjugation from inducing the SOS response (44). Although the gene has
not been identified, the isfA mutation of E. coli
also has been found to suppress RecA coprotease-dependent
cleavage of UmuD (45). Finally, overexpression of the DinI protein of
E. coli inhibits both the coprotease and recombinase
activities of RecA in vivo (5). A dinI mutant
showed no decrease in UV resistance but exhibited increased cleavage of
UmuD and higher SOS mutagenesis than the parent strain, suggesting that
DinI may act specifically to down-regulate SOS mutagenesis (5). DinI
also inhibits RecA activity in vitro but only when present
in vast molar excess (17- to 30-fold) of RecA (5, 40). In contrast,
E. coli RecX:RecA molar ratios of 1:44 and 1:70, respectively, were sufficient for complete inhibition of RecA-promoted DNA strand exchange and ATPase activity. Thus, RecX appears to be a
stronger inhibitor of RecA activity than DinI in vitro. It is likely that a complicated network of interactions between the RecX,
DinI, LexA, UmuD, SSB, RecA, and possibly other unidentified proteins,
acts to regulate the RecA nucleoprotein filament and RecA function.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(Invitrogen), BL21(DE3) (Novagen),
AB1157 (18), AB1157
recA (19), Hfr Cavalli (20),
DE192(lexA51) (R. Woodgate),
DM49(lexA3) (21),
sulA::lacZ'YA::kan from strain SY2 (22), and
dinD1::Mud(AmpR,
lacZ'YA) from strain JH39 (23). All strains used
in UmuD studies additionally contained umuD on a low copy
number plasmid (pRW362) (24). Plasmids pKD4, pKD46, and pCP20 (25) were
used for creation of a recX deletion strain. Media for
E. coli were prepared as described previously (17). All
media and plasmids used for yeast two-hybrid analyses were from
Invitrogen. Antibiotics were added at the following concentrations:
ampicillin (Amp), 100 µg/ml; kanamycin (Kan), 15 µg/ml for
chromosomal markers, or 40 µg/ml for plasmid markers; spectinomycin,
50 µg/ml; streptomycin, 50 µg/ml; erythromycin (Erm), 250 µg/ml;
chloramphenicol, 100 µg/ml. Isopropyl
-D-thiogalactopyranoside (IPTG) (Diagnostic Chemicals Ltd.) was used at 1 or 1.5 mM, mitomycin C (MMC) (Sigma)
was used at 1 or 0.2 µg/ml, and
5-bromo-4-chloro-3-indolyl-
-D-galactopyranoside (X-
-gal) (Clontech) was used at 20 µg/ml. ATP was from Amersham Biosciences.
recX resulted in similar
levels of UV resistance, demonstrating the activity of HisRecX in
vivo (data not shown).
recX::kan. PCR analysis of
KanR colonies using primer pairs ECRX1R
(5'-GCTGGTAACTGAAAAGTGGG-3') and ECRX2F (5'-AGGCGTAGCAGAAACTAACG-3'),
just outside the recX coding region, and
kan-specific primers k1 and k2 (25) confirmed the location
and insertion of the kan gene (data not shown). The subsequent eviction of the kan gene from strain
AB1157
recX::kan, using a curable
helper plasmid encoding the FLP recombinase (pCP20), yielding
strain AB1157
recX, was verified by PCR (data not shown).
recX, t0
aliquots were taken, both cultures were exposed to 4 J/m2,
and additional aliquots were taken after 2, 4, 6, 8, 10, 12, 15, 20, and 30 min. Protein concentration of samples was determined by BCA
assay (Pierce).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-gal, indicating both MEL1 and
HIS3 reporter gene expression in these cells (Fig.
1 and data not shown). However, cells
grew less well on media lacking adenine, histidine, tryptophan, and leucine, suggesting lower expression of the ADE2 reporter
gene, which demands strong protein-protein interactions for its
expression (data not shown). AH109 cells carrying control plasmids with
interacting gene products (pGADT7-T and pGBKT7-53) grew on media
lacking histidine, tryptophan, and leucine, but AH109 carrying control
plasmids with non-interacting gene products (pGADT7-T and pGBKT7-Lam),
or AH109 co-transformed with the recA or recX
plasmids and either pGADT7-T or pGBKT7-Lam plasmids did not grow on the
selective media (Fig. 1 and data not shown). Together these data
demonstrate a specific interaction of E. coli RecA and RecX
proteins in yeast.
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Fig. 1.
Yeast two-hybrid analysis of RecX and RecA
interaction. Yeast AH109 cells containing various constructs of
pGBT7 and pGADT7 grown at 30 °C on complete synthetic media lacking
tryptophan, leucine, and histidine. Each quadrant contains yeast cells
streaked from a single transformant. Top half of plate,
left to right are the negative
(pGADT7-T/pGBKT7-Lam) and positive (pGADT7-T/pGBKT7-53) controls;
bottom half, left to right are
pGADT7-RecA/pGBKT7-RecX and pGADT7-RecX/pGBKT7-RecA.
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Fig. 2.
Effect of RecX on RecA-promoted DNA strand
exchange. Position of bands corresponding to ssDNA
(SS), linearized dsDNA (L), nicked circular dsDNA
(NC), and joint molecules (JM), are indicated.
Time course of RecA-promoted DNA strand exchange in absence
(lanes 1-5, RecX:RecA molar ratio 1:2.2) or presence
(lanes 6-10) of RecX and titration of RecX inhibition of
RecA-promoted DNA strand exchange over 30 min (lanes
11-18). Control lanes 11 and 18 contain no
RecX, no RecA or RecX, respectively. Molar ratios of RecX:RecA for
lanes 12-17 are as follows: 1:22, 1:44, 1:88, 1:177, 1:354,
and 1:707.
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Fig. 3.
Effect of RecX on ssDNA-dependent
ATP hydrolysis by RecA protein in the presence and absence of SSB.
Rates of hydrolysis are expressed as the percentage of control
reactions without RecX. Molar ratios of RecX:RecA are as follows: 1:70
(60 nM RecX), 1:42 (100 nM RecX), 1:14 (300 nM RecX), 1:8 (500 nM RecX), and 1:4 (1000 nM RecX).
recX mutant, demonstrating that recX is
induced upon DNA damage (Fig. 4). Robust expression of the ~19-kDa
RecX band was detected in strain DE192, a lexA51(Def) strain
that does not produce a functional LexA repressor, but not in the
isogenic strain DE192
recX (data not shown). Moreover, RecX levels did not increase after addition of MMC to cultures of
DM49(lexA3), which has a non-cleavable LexA repressor (data not shown). Finally, RecA and RecX proteins showed identical patterns of induction after UV treatment (data not shown), as was also found
with recA and recX transcripts (31), and we
demonstrated that recA and recX are present on
the same transcript by RT-PCR (data not shown). Together these data
demonstrate that recX is induced with recA in a
LexA-dependent manner and is, therefore, an SOS response
gene.
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Fig. 4.
Western blot time course of RecX
expression. Cells were grown to A600 = 0.4 (min 0), and basal or MMC (1 µg/ml)-induced RecX expression was
monitored over time in strain AB1157. AB1157 recX is
included as a negative control. 10 µg of total protein was loaded per
lane, blotted, and developed using polyclonal anti-RecX antisera.
recX mutation on UV resistance in
strain AB1157. Strain AB1157
recX showed a small, but
statistically significant, decrease in UV resistance relative to AB1157
(Fig. 5A). Because the
recX mutation is non-polar (see "Experimental
Procedures"), this phenotype is due to recX inactivation.
To test this assumption, a functional copy of recX was
introduced in trans (pGCC4/recX), with
recX under control of lac regulatory elements,
into strain AB1157
recX. Surprisingly, this plasmid
conferred a striking reduction in UV resistance relative to strain
AB1157
recX carrying pGCC4 alone (Fig. 5B).
Titrating the amount of IPTG in the growth medium, we observed that
increasing levels of IPTG induction resulted in decreased UV
resistance, suggesting that the amount of RecX protein produced was
affecting UV resistance (data not shown).
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Fig. 5.
UV resistance of
recX mutant or overexpression strains.
A, relative survival of AB1157 and
recX
mutant. B, AB1157
recX carrying pGCC4 (vector)
or pGCC4/recX. IPTG (1.5 mM) was added to both
cultures. Error bars represent the standard error of the
mean of at least two independent experiments done in duplicate.
Differences between strains AB1157 and
recX are
statistically significant at all UV doses (excluding 10 J) at
p < 0.01 by the Student's t test.
Differences between strain AB1157
recX carrying pGCC4 or
pGCC4/recX are statistically significant at all UV doses at
p < 0.05 by the Student's t test.
-galactosidase activity. After addition of MMC, cells
carrying pGCC4/recX showed statistically significant lower
induction of both sulA (3- to 4-fold; Fig.
6A) and dinD1 (data
not shown) relative to those carrying the vector alone. Therefore,
overexpression of RecX inhibits induction of the SOS response. To
determine whether a
recX strain would show increased SOS
induction, the sulA::lacZ fusion was transduced into strains AB1157 and AB1157
recX to yield
strains ES1 and ES2, respectively. After incubation with MMC (data not shown), or exposure to UV light (Fig. 6B), strain ES2
(
recX) showed the same increase in sulA
expression as strain ES1. These results suggest that the chromosomal
recX does not affect SOS induction.
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Fig. 6.
sulA::lacZ
induction in recX mutant or overexpression
strain. A, strain SY2
(sulA::lacZ'YA::kan)
carrying pGCC4 (vector) or pGCC4/recX was grown in LB-Erm
(1.5 mM IPTG) to A600 ~ 0.2, MMC
(0.2 µg/ml) was added, and samples were taken and assayed for
-galactosidase activity (17). Error bars represent the
mean of at least two independent experiments done in duplicate.
Differences between strains are statistically significant at all time
points at p < 0.01 by the Student's t
test. B, strains ES1 and ES2 (
recX),
containing a chromosomal sulA::lacZ
fusion, were grown in LB with 0.1% glucose to
A600 ~ 0.4 and exposed to 4 J/m2
UV light, and samples were taken and assayed for
-galactosidase
activity. Error bars represent the mean of three independent
experiments.
recX
(data not shown). Strain AB1157
recX carrying
pGCC4/recX (induced with IPTG) showed about 10% less RecA
than cells carrying pGCC4 (data not shown), but these differences are
too small to account for the large effect on SOS induction and UV
resistance and suggest that the inhibitory effects of RecX are largely
due to effects on RecA activity. Further experimental support for a
direct and specific effect of RecX on RecA activity in vivo
came from UV resistance studies in a recA deletion strain.
The UV resistance of strain AB1157
recA carrying plasmids
pGCC4 or pGCC4/recX (induced with IPTG) was the same (data
not shown). Together these data strongly suggest that the phenotypes
observed in cells overexpressing RecX are due to effects on RecA
activity, not RecA levels. Moreover, these effects appear to be
specifically mediated through RecA, an observation that is further
supported by the interaction of RecX and RecA in a yeast two-hybrid
assay, and are not simply due to some artifact of RecX protein overexpression.
recX (data not
shown).
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Fig. 7.
LexA and UmuD processing in RecX
overexpression strains. A, representative Western blot
of LexA levels in strain AB1157 recX carrying pGCC4
(vector) or pGCC4/recX (both with 1.5 mM IPTG)
at times indicated after exposure to 8 J/m2 UV irradiation.
6 µg of total protein per lane was loaded for subsequent Western blot
analysis using anti-LexA antisera. B, representative Western
blot of UmuD levels in strain DE192
recX carrying pRW362
(umuD) and either pGCC4 or pGCC4/recX (both with
1.5 mM IPTG) at times indicated after addition of MMC (0.2 µg/ml). 60 µg of total protein was loaded per lane for subsequent
Western blot analysis using anti-UmuD antisera.
recX (data not shown). Taken together, these results
demonstrate that RecX overexpression inhibits the coprotease activity
of RecA; however, the chromosomal recX does not have a
measurable effect on coprotease activity in vivo.
recX (Fig.
8; data not shown). However, the P1
transduction frequency of both markers was significantly reduced in
AB1157
recX cells carrying plasmids pGCC4/HisRecX
(>100-fold reduction) (Fig. 8; data not shown) or
pGCC4/recX (data not shown) relative to those carrying the
pGCC4 vector (Fig. 8; data not shown). Strain AB1157
recX
carrying pGCC4/recX also showed decreased Hfr conjugation
relative to the pGCC4 vector control strain (data not shown). These
results indicate that RecX can inhibit RecA recombinase activity
in vivo and support our observations that RecX inhibits DNA
strand exchange in vitro.
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Fig. 8.
P1 transduction frequency of
recX or HisRecX overexpression strains.
Generalized transduction frequency of proline prototrophic marker in
strains AB1157, AB1157
recX, and AB1157
recX
carrying plasmids pGCC4 or pGCC4/HisRecX. Error bars
represent the mean of three experiments done in triplicate (AB1157 and
recX) or the mean of two experiments done in duplicate
(+pGCC4 and +pGCC4/HisRecX).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
recX mutant was a small decrease in UV resistance.
Therefore, one hypothesis is that RecX functions in DNA repair.
Accordingly, we observed no differences in recombination ability, as
measured by P1 transduction or Hfr conjugation (Fig. 8 and data not
shown) between the
recX mutant and the parent strain.
This is probably due to the exceedingly low basal levels of RecX, less
than 50 molecules per cell. Using semi-quantitative immunoblotting, we
calculated the basal level of RecA molecules to be ~15,000 molecules
per cell (data not shown), which is consistent with previous reports
(37, 38). Therefore, although RecX can completely abolish pairing of
homologous DNA molecules (joint molecule formation) in vitro
at a RecX:RecA molar ratio of 1:44, the RecX:RecA molar ratio inside
the bacterial cell is significantly below this, at most 1:300. DNA
damage resulted in increased levels of recX transcript in
E. coli and other bacteria (11, 12, 15, 31, 39). In our
studies, after treating E. coli with MMC, RecX and RecA
protein levels increased to ~800 and 100,000 molecules per cell,
respectively (data not shown), so the RecX:RecA ratio is 1:125, which
is closer to the level where we saw inhibition of joint molecule
formation in vitro. Therefore we propose that the biological
role of RecX is manifest during the SOS response. It is possible that
some threshold level of RecX is reached or that additional factors
influence the ability of RecX and RecA to interact under these
circumstances, resulting in the observed phenotype of decreased UV
resistance. In support of this hypothesis, the DinI protein, which also
modulates RecA activity, showed increased affinity for the RecA protein
in vivo at later stages of the SOS response (40). Moreover,
because recX is directly downstream of and co-transcribed
with recA, RecX and RecA are likely to be translated in the
same region of the E. coli cell, allowing the local
intracellular concentration of RecX to be higher, possibly driving
interaction with the RecA protein.
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ACKNOWLEDGEMENTS |
---|
We thank A. Criss, K. Kline, and D. Tobiason for editorial suggestions for the manuscript. We also thank M. Cox for providing antibodies, P. Model and H. Ohmori for providing strains, and R. Woodgate for generously providing antibodies, strains, and technical assistance.
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FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grants RO1-AI44239 (to H. S. S.) and RO1-GM-62653 (to S. C. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by American Cancer Society postdoctoral fellowship PF-00-016-01-GMMC.
Present address: Molecular Staging, Inc., 300 George St.,
Suite 701, New Haven, CT 06511.
** Present address: Dept. of Microbiology and Immunology, Loyola University Medical Center, Maywood, IL 60153.
To whom correspondence should be addressed: Dept. of
Microbiology and Immunology, Feinberg School of Medicine, Northwestern University, 303 E. Chicago Ave., Chicago, IL 60611. Tel.: 312-503-9788; Fax: 312-503-1339; E-mail: h-seifert@northwestern.edu.
Published, JBC Papers in Press, November 9, 2002, DOI 10.1074/jbc.M210496200
2 R. D. Porter, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are:
ssDNA, single-strand
DNA;
dsDNA, double-strand DNA;
IPTG, isopropyl
-D-thiogalactopyranoside;
Kan, kanamycin;
Erm, erythromycin;
MMC, mitomycin C;
HA, hemagglutinin;
SSB, single-stranded
DNA binding.
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