Single-Strand-Specific Exonucleases Prevent Frameshift Mutagenesis by Suppressing SOS Induction and the Action of DinB/DNA Polymerase IV in
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《细菌学杂志》
Departments of Molecular and Human Genetics,Biochemistry and Molecular Biology and Molecular Virology and Microbiology, Baylor College of Medicine, One Baylor Plaza, Room S809A Mail Stop BCM225, Houston, Texas 77030-3411
ABSTRACT
Escherichia coli strains carrying null alleles of genes encoding single-strand-specific exonucleases ExoI and ExoVII display elevated frameshift mutation rates but not base substitution mutation rates. We characterized increased spontaneous frameshift mutation in ExoI– ExoVII– cells and report that some of this effect requires RecA, an inducible SOS DNA damage response, and the low-fidelity, SOS-induced DNA polymerase DinB/PolIV, which makes frameshift mutations preferentially. We also find that SOS is induced in ExoI– ExoVII– cells. The data imply a role for the single-stranded exonucleases in guarding the genome against mutagenesis by removing excess single-stranded DNA that, if left, leads to SOS induction and PolIV-dependent mutagenesis. Previous results implicated PolIV in E. coli mutagenesis specifically during starvation or antibiotic stresses. Our data imply that PolIV can also promote mutation in growing cells under genome stress due to excess single-stranded DNA.
INTRODUCTION
Escherichia coli possesses at least four single-strand-specific exonucleases that function in DNA repair. ExoI and ExoX are 3' end specific (22, 45), RecJ is 5' end specific (24), and ExoVII degrades single-strand ends of either polarity (4). Despite their differing polarities, these exonucleases are wholly or partially redundant for their functions in vivo. Single-strand-specific exonucleases of either 5' or 3' polarity are required for normal levels of homologous recombination (30, 35, 46) and presumably recombinational repair. In their absence, recombination of linear substrates is reduced (30, 35, 46), suggesting that processing of double-strand ends to yield either 3' or 5' single-strand overhangs promotes recombination (35, 37, 40).
In the E. coli methyl-directed mismatch repair system, single-strand-specific exonucleases are required for degradation of the nicked, error-containing strand in vitro (44). The polarity of the exonuclease required depends on the location of the incision relative to the mismatch, and any of the four semiredundant single-strand-specific exonucleases is sufficient (44). Likewise, in vivo, these exonucleases are redundant for mismatch repair, which remains functional until all four exonucleases are lost (14, 44, 46). Loss of all four causes a mutator phenotype from the inability to repair both frameshift and base substitution errors (44).
Interestingly, E. coli strains deficient for only two or three of the single-strand-dependent exonucleases (ExoI and ExoVII or ExoI, ExoVII, and RecJ) display a frameshift-biased mutator phenotype (46) with little effect on base-substitution mutation rates (14, 44, 46). ExoI– ExoVII– RecJ– cells showed increased spontaneous frameshift mutation rates, which were attributed to loss of ExoI and ExoVII (46). In ExoI- ExoVII-deficient cells, induced frameshift mutations were also increased (46). One hypothesis to explain the frameshift bias of the mutator phenotype in ExoI- ExoVII-deficient cells is that the 3' single-strand-exonuclease activities degrade frameshift strand-slippage intermediates before, as well as during, mismatch repair (46). In their absence, frameshift errors could be more likely to persist.
We considered, and report data that support, an alternative hypothesis: that the frameshift-mutator phenotype of ExoI– ExoVII– cells could result from enhanced SOS induction promoted by the presence of excess single-stranded DNA (ssDNA) in cells. ssDNA is the signal that induces the SOS DNA damage response (39), which leads to derepression of more than 40 damage response genes (6, 18). Included among these is dinB (19), encoding DNA polymerase IV (PolIV, or DinB) (47). PolIV is a poorly processive, low-fidelity DNA polymerase that preferentially makes –1 frameshift errors and, to a lesser extent, single-base substitutions (42, 47) and is a member of the Y family of DNA polymerases (33). PolIV promotes untargeted mutagenesis of lambda phage in SOS-induced cells (1, 19, 50). Overexpression of dinB from a plasmid also increases frameshift and some base-substitution mutations without exogenous DNA-damaging agents (19, 48). PolIV is also required for stationary-phase, stress-induced mutagenesis at episomal (8, 27) and chromosomal (2) sites and for chromosomal mutations during antibiotic stress (5), but not normally for spontaneous frameshift or substitution mutagenesis in rapidly growing cells (21, 27, 29, 49). For stationary-phase mutagenesis, PolIV generates mutations in acts of double-strand break repair, which switches to a low-fidelity mode using PolIV under the control of the RpoS general stress response (34). We hypothesized that upon SOS induction in ExoI– ExoVII– cells, PolIV might compete with the major replicative polymerase (PolIII), resulting in PolIV-mediated mutagenesis during growth. A role for PolIV could explain the frameshift bias of the ExoI– ExoVII– mutator phenotype. We provide support for this hypothesis here. We find that ExoI– ExoVII– cells are SOS induced and that the increase in spontaneous frameshift mutation in ExoI- ExoVII-defective cells requires RecA, an inducible LexA/SOS repressor, and PolIV. The data indicate an important role for the single-strand-specific exonucleases in guarding the genome against SOS induction and consequent mutagenesis.
MATERIALS AND METHODS
Bacterial strains and media. Isogenic bacterial strains used in this study are shown in Table 1. These were constructed by bacteriophage P1-mediated transduction as described previously (31). Antibiotics were used at the following concentrations (μg/ml): ampicillin, 100; chloramphenicol, 25; kanamycin, 30; tetracycline, 10; and 5-fluorocytosine, 20. All M9 media (31) also contained vitamin B1 (10 μg/ml) and either 0.1% glycerol or 0.1% lactose. Luria-Bertani-Herskowitz (LBH) medium was described previously (43).
lac frameshift-reversion assay. We measured spontaneous reversion of an F'-borne lac +1 frameshift allele in growing cells in 15- to 40-tube fluctuation tests, using methods that avoid inclusion in the data of stress-induced "adaptive" Lac+ mutants that form during selection for Lac+ revertants on lactose medium (the methods are reviewed in references 12 and 29). Strains carrying the frameshift allele ("testers"), and also Lac+ revertant control strains (8 to 12 independent spontaneous Lac+ revertants for each strain), were grown into single colonies, which were used to inoculate multiple independent cultures for fluctuation tests. For each culture, a single colony was resuspended in 5 ml of M9 glycerol liquid medium and grown to saturation (2 days). Tester cells were washed and resuspended in M9 buffer. Lac+ controls were diluted in M9 buffer to 1,000 cells/ml. Both testers and controls were mixed with concentrated (25x) FC29 "scavenger" cells (3) (not Lac revertible) in top agar and plated on M9 lactose medium; scavengers prevent the growth of frameshift-bearing cells on lactose selection plates by scavenging any nonlactose carbon sources. The plates were incubated at 37°C. Newly visible Lac+ colonies were scored every 2 hours, and control plates with a known dilution of Lac+ control strains were used to calculate the time at which 50% of the colonies were visible (T50) for each genotype. To avoid the risk of counting adaptive Lac+ mutants (formed after selection) (3, 28), mutants were counted at the T50 of their respective Lac+ parental strains (29), and the counts were extrapolated to estimate the number at T100. (If T50 was missed, time points between T40 and T66 were used.) The extrapolated numbers at T100 were used to determine the numbers of generation-dependent (preplating) Lac+ mutant cells per culture. Total CFU were assayed on LBH plates. Mutation rates were estimated from these mutant frequencies by the MMS maximum likelihood method (36).
Chromosomal Tet frameshift-reversion assay. Strains carrying a +1 frameshift allele of the Tn10 tetA gene disrupting the chromosomal upp gene (2) were monitored for reversion to tetracycline resistance (Tetr) in 11- to 19-tube fluctuation tests similar to those described for Lac (above) but without scavenger cells. Cells were plated on LBH with and without tetracycline and incubated at 37°C, Tetr and total colonies were scored at 48 h, and mutation rates were estimated as described above.
Measurement of SOS induction. Exonuclease-deficient and isogenic Exo+ cells carrying a chromosomal gfp reporter under the control of the sulA promoter (15, 25) were grown to saturation in M9 glycerol, concentrated, and photographed under the microscope (15) using fluorescence detection (for green fluorescent protein [GFP]) and bright field for the same fields of view. At least 12 fields were photographed per strain each day for quantification of highly GFP-positive cells.
RESULTS AND DISCUSSION
Increased spontaneous frameshift mutation rate in ExoI– ExoVII– cells at chromosomal and episomal sites. To study the frameshift mutator phenotype of exonuclease-deficient strains (46), we constructed ExoI– ExoVII– (xonA xseA) derivatives of two different +1 frameshift reporter strains for mutation rate determinations. The first carries the lacI33lacZ fusion allele on an F' episome (3) in which reversion to Lac+ can occur by a net –1 frameshift mutation within a 128-basepair region (10, 38). The second carries a chromosomal +1 frameshift allele of the tetA gene (upp::Tn10dtet+1 [2]), which confers tetracycline resistance (Tetr) upon –1 deletion at a run of five Gs (2, 9). The mutation rates in single-strand-exonuclease-proficient (Exo+) cells were 9.3 (±2) x 10–10 and 2.6 (±0.2) x 10–9 mutations per cell per generation for the episomal lac and chromosomal tet alleles, respectively (Tables 2 and 3).
We found that deletion of xonA (encoding ExoI) and xseA (encoding the large subunit of ExoVII) increased the spontaneous frameshift-mutation rates significantly: an average of 10-fold (±2-fold) for the episomal-lac reporter and 20-fold (±3-fold) for the chromosomal tet reporter when rates were compared within each experiment (Tables 2 and 3 and Fig. 1A). The data imply that an impaired ability to degrade ssDNA results in increased frameshift mutation at both chromosomal and episomal loci. Previous identification of the frameshift-mutator phenotype of cells lacking ExoI and ExoVII used assays of mutation at episomal sites (46). Those findings are confirmed and generalized here.
SOS induction in ExoI– ExoVII– cells. We used a gfp reporter gene under the control of the LexA-repressed sulA promoter to quantify GFP-positive cells as a function of SOS/LexA-regulon derepression, using fluorescence microscopy (15, 25). A previous study using this system to assay SOS induction found that mutations that lead to SOS induction can do so either in all cells or by increasing the size of a subpopulation of spontaneously green cells, or both, and both are dependent on RecA (25). The ExoI– ExoVII– strain showed increases in both kinds of induction. First, most cells appeared slightly greener than those of the isogenic Exo+ strain (Fig. 1B to E), and second, the subpopulation of very green cells was larger; the very green cells were 4.2% (81/1,922) of all cells in the ExoI– ExoVII– strain and 1.1% (22/1,937) of the isogenic Exo+ strain (data collected on two different days; P < 0.001 by z test [SigmaStat 3.1; Systat Software, Inc., Point Richmond, CA]). The greenness is RecA dependent, and thus, genuine SOS induction (25; J. M. Pennington and S. M. Rosenberg, unpublished data; and data not shown). Thus, SOS is induced in ExoI– ExoVII– cells. These data imply a role for these exonucleases in preventing/reducing spontaneous SOS induction by removal of excess ssDNA. The ssDNA normally removed by ExoI and ExoVII appears to arise at low levels in most cells, perhaps from normal DNA metabolic processes, and at higher levels/more persistently in rare cells, perhaps from sporadic spontaneous DNA damage.
Hypermutation in ExoI– ExoVII– cells requires the SOS response. The SOS DNA damage response is activated when RecA binds ssDNA, forming a nucleoprotein filament in which RecA is activated (called RecA) for facilitating autoproteolytic cleavage of target SOS response proteins, including the LexA transcriptional repressor (11). Thus, induction of SOS/LexA-controlled genes occurs neither in cells that lack RecA nor in cells that carry a special mutant allele of lexA, lexA(Ind–), that encodes an uncleavable repressor (23, 32). We observed that the increased Lac+ and Tetr frameshift mutation rates seen in ExoI– ExoVII– cells were greatly reduced in strains that also lack RecA or carry the lexA(Ind–) allele (Tables 2 and 3 and Fig. 1A). ExoI– ExoVII– RecA– cells had five- to sevenfold-lower mutation rates than isogenic ExoI– ExoVII– RecA+ controls (Fig. 1A). Similarly, the presence of the lexA(Ind–) allele decreased mutation rates three- to fourfold relative to isogenic LexA+ controls (Fig. 1A). The ExoI– ExoVII– RecA– and ExoI– ExoVII– LexA(Ind–) strains show mutation rates only slightly, and not significantly, greater than those of their respective Exo+ parent strains (Tables 2 and 3 and Fig. 1A), indicating that most of the hypermutation in ExoI– ExoVII– cells requires SOS induction.
Hypermutation in ExoI– ExoVII– cells requires DNA polymerase IV. We found that increased frameshift mutation in ExoI– ExoVII– cells required the SOS-upregulated DinB/PolIV DNA polymerase (Tables 2 and 3 and Fig. 1A). We used ExoI– ExoVII– cells that carry the dinB10 allele, which encodes a catalytically inactive enzyme (47) but is not polar on downstream genes in the dinB operon (29). dinB10 reduced the frameshift mutation rates to levels near those in Exo+ cells. Three- to 22-fold decreases relative to the isogenic ExoI– ExoVII– strains were seen, depending on the assay (Fig. 1A). In both frameshift reversion assays, the ExoI– ExoVII– PolIV– mutation rates were not significantly different from those of the respective Exo+ parent strains (Tables 2 and 3). Thus, hyper-frameshift mutation in ExoI– ExoVII– cells requires a catalytically active PolIV, implicating PolIV as the SOS-controlled component required.
Further discussion. The data reported show that E. coli strains deficient for ExoI and ExoVII single-strand-dependent exonucleases exhibit increased spontaneous frameshift mutation rates at F' episomal and chromosomal sites, as shown previously for an episomal locus (46). This increase requires RecA, a cleavable LexA/SOS repressor, and DNA PolIV (Tables 2 and 3 and Fig. 1A). The absence of ExoI and ExoVII does not diminish DNA mismatch repair activity (14, 44, 46). However, DNA metabolism is altered sufficiently to provoke SOS induction (Fig. 1B to E and see above). We note that previous analyses of the same Lac and Tet mutation assays in the Exo+ genetic background showed no PolIV-dependent (27, 29) or RecA-dependent (2, 13) spontaneous mutation; it is specific to the ExoI- ExoVII-defective background, implying that the enhanced SOS induction in ExoI- ExoVII-defective cells is responsible.
In Fig. 2, we suggest that excess linear ssDNAs are normally removed by ExoI and ExoVII, and their absence leads to SOS induction, PolIV up-regulation, and PolIV-dependent mutagenesis. PolIV might then more often displace the replicative DNA PolIII either during acts of normal DNA replication or during acts of replication in repair of spontaneous DNA double-strand breaks (34). The ssDNA ends normally removed by ExoI and ExoVII might arise during normal DNA metabolic processes, such as replication or repair, during sporadic DNA damage and repair events, or both.
The increase in the spontaneous frameshift mutation rate due to disruption of ExoI and ExoVII is less pronounced in a MG1655 or JC11450 background (data not shown) than with FC40, used here, which carries F'128. This is likely to be due to the lower level of dinB expression in those backgrounds, which lack the higher-expression episomal copy of dinB in F'128 (20). Because the initial description of increased episomal frameshift mutation in the absence of ExoI and ExoVII was also done with F'128 (46), our data are relevant to those data. Whereas the mechanism of frameshift mutation suppression by ExoI and ExoVII in that assay might have been solely via editing of frameshift mutation intermediates (46), our data indicate that an additional mechanism of suppression, by suppressing SOS induction, is also important, and in the F'128 background it appears to dominate. It is possible that some of the increase in mutation, even in this genetic background, may occur via lack of direct editing of frameshift intermediates, as proposed previously (46), because although the recA, lexA(Ind–), and dinB derivatives do not show significant differences from the Exo+ strain, they might have more mutations than Exo+ (Tables 2 and 3 and Fig. 1A).
Another low-fidelity DNA polymerase, DNA PolV, is also upregulated during SOS but is unlikely to be relevant to spontaneous mutation in ExoI– ExoVII– cells because PolV provokes substitution mutations preferentially (41), and these are not increased appreciably (14, 46). PolV becomes active only late in severe SOS responses (41). We observed only low-level SOS induction in most ExoI– ExoVII– cells (Fig. 1B to E), which may be insufficient for PolV induction, although it is mutagenic via PolIV.
Although fully redundant for mismatch repair activity (14, 44, 46), the four single-strand-specific exonucleases appear to be only partially redundant for maintenance of genomic stability. First, defects in recombination are seen when two or three are defective (30, 35, 46). Second, we show here that lack of even two can be mutagenic via increased SOS induction and PolIV up-regulation. The mutation mechanism at least partially resembles starvation stress-induced adaptive mutation in the E. coli Lac system (17) and antibiotic-induced resistance mutation (5) in that cells under starvation or antibiotic stress have increased mutation rates, depending on LexA-regulon derepression and PolIV (2, 5, 8, 26, 27). Previously, PolIV-dependent mutagenesis of E. coli was observed exclusively under conditions of starvation stress (2, 8, 27) or antibiotic stress (5) or during artificial PolIV overproduction (19, 48) and not spontaneously in growing cells (29, 49), unless the starvation stress response was artificially expressed (34). Here, the stress that promotes mutation is genomic stress, apparently from insufficient cleansing of SOS-provoking linear ssDNAs. The single-strand-specific exonucleases thus function as guardians of the genome, suppressing up-regulation of SOS genes and consequent mutagenesis.
ACKNOWLEDGMENTS
We thank Daniel B. Magner for assistance with computational aspects of data analysis; Mellanie P. Ray for technical assistance; Robert Do, Gbenga Olanrewaju, and Kenny Tran for preparation of media; and Matthew D. Blankschien, Janet L. Gibson, P. J. Hastings, Christophe Herman, Mary-Jane Lombardo, and Jeanine M. Pennington for helpful discussions and comments on the manuscript.
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ABSTRACT
Escherichia coli strains carrying null alleles of genes encoding single-strand-specific exonucleases ExoI and ExoVII display elevated frameshift mutation rates but not base substitution mutation rates. We characterized increased spontaneous frameshift mutation in ExoI– ExoVII– cells and report that some of this effect requires RecA, an inducible SOS DNA damage response, and the low-fidelity, SOS-induced DNA polymerase DinB/PolIV, which makes frameshift mutations preferentially. We also find that SOS is induced in ExoI– ExoVII– cells. The data imply a role for the single-stranded exonucleases in guarding the genome against mutagenesis by removing excess single-stranded DNA that, if left, leads to SOS induction and PolIV-dependent mutagenesis. Previous results implicated PolIV in E. coli mutagenesis specifically during starvation or antibiotic stresses. Our data imply that PolIV can also promote mutation in growing cells under genome stress due to excess single-stranded DNA.
INTRODUCTION
Escherichia coli possesses at least four single-strand-specific exonucleases that function in DNA repair. ExoI and ExoX are 3' end specific (22, 45), RecJ is 5' end specific (24), and ExoVII degrades single-strand ends of either polarity (4). Despite their differing polarities, these exonucleases are wholly or partially redundant for their functions in vivo. Single-strand-specific exonucleases of either 5' or 3' polarity are required for normal levels of homologous recombination (30, 35, 46) and presumably recombinational repair. In their absence, recombination of linear substrates is reduced (30, 35, 46), suggesting that processing of double-strand ends to yield either 3' or 5' single-strand overhangs promotes recombination (35, 37, 40).
In the E. coli methyl-directed mismatch repair system, single-strand-specific exonucleases are required for degradation of the nicked, error-containing strand in vitro (44). The polarity of the exonuclease required depends on the location of the incision relative to the mismatch, and any of the four semiredundant single-strand-specific exonucleases is sufficient (44). Likewise, in vivo, these exonucleases are redundant for mismatch repair, which remains functional until all four exonucleases are lost (14, 44, 46). Loss of all four causes a mutator phenotype from the inability to repair both frameshift and base substitution errors (44).
Interestingly, E. coli strains deficient for only two or three of the single-strand-dependent exonucleases (ExoI and ExoVII or ExoI, ExoVII, and RecJ) display a frameshift-biased mutator phenotype (46) with little effect on base-substitution mutation rates (14, 44, 46). ExoI– ExoVII– RecJ– cells showed increased spontaneous frameshift mutation rates, which were attributed to loss of ExoI and ExoVII (46). In ExoI- ExoVII-deficient cells, induced frameshift mutations were also increased (46). One hypothesis to explain the frameshift bias of the mutator phenotype in ExoI- ExoVII-deficient cells is that the 3' single-strand-exonuclease activities degrade frameshift strand-slippage intermediates before, as well as during, mismatch repair (46). In their absence, frameshift errors could be more likely to persist.
We considered, and report data that support, an alternative hypothesis: that the frameshift-mutator phenotype of ExoI– ExoVII– cells could result from enhanced SOS induction promoted by the presence of excess single-stranded DNA (ssDNA) in cells. ssDNA is the signal that induces the SOS DNA damage response (39), which leads to derepression of more than 40 damage response genes (6, 18). Included among these is dinB (19), encoding DNA polymerase IV (PolIV, or DinB) (47). PolIV is a poorly processive, low-fidelity DNA polymerase that preferentially makes –1 frameshift errors and, to a lesser extent, single-base substitutions (42, 47) and is a member of the Y family of DNA polymerases (33). PolIV promotes untargeted mutagenesis of lambda phage in SOS-induced cells (1, 19, 50). Overexpression of dinB from a plasmid also increases frameshift and some base-substitution mutations without exogenous DNA-damaging agents (19, 48). PolIV is also required for stationary-phase, stress-induced mutagenesis at episomal (8, 27) and chromosomal (2) sites and for chromosomal mutations during antibiotic stress (5), but not normally for spontaneous frameshift or substitution mutagenesis in rapidly growing cells (21, 27, 29, 49). For stationary-phase mutagenesis, PolIV generates mutations in acts of double-strand break repair, which switches to a low-fidelity mode using PolIV under the control of the RpoS general stress response (34). We hypothesized that upon SOS induction in ExoI– ExoVII– cells, PolIV might compete with the major replicative polymerase (PolIII), resulting in PolIV-mediated mutagenesis during growth. A role for PolIV could explain the frameshift bias of the ExoI– ExoVII– mutator phenotype. We provide support for this hypothesis here. We find that ExoI– ExoVII– cells are SOS induced and that the increase in spontaneous frameshift mutation in ExoI- ExoVII-defective cells requires RecA, an inducible LexA/SOS repressor, and PolIV. The data indicate an important role for the single-strand-specific exonucleases in guarding the genome against SOS induction and consequent mutagenesis.
MATERIALS AND METHODS
Bacterial strains and media. Isogenic bacterial strains used in this study are shown in Table 1. These were constructed by bacteriophage P1-mediated transduction as described previously (31). Antibiotics were used at the following concentrations (μg/ml): ampicillin, 100; chloramphenicol, 25; kanamycin, 30; tetracycline, 10; and 5-fluorocytosine, 20. All M9 media (31) also contained vitamin B1 (10 μg/ml) and either 0.1% glycerol or 0.1% lactose. Luria-Bertani-Herskowitz (LBH) medium was described previously (43).
lac frameshift-reversion assay. We measured spontaneous reversion of an F'-borne lac +1 frameshift allele in growing cells in 15- to 40-tube fluctuation tests, using methods that avoid inclusion in the data of stress-induced "adaptive" Lac+ mutants that form during selection for Lac+ revertants on lactose medium (the methods are reviewed in references 12 and 29). Strains carrying the frameshift allele ("testers"), and also Lac+ revertant control strains (8 to 12 independent spontaneous Lac+ revertants for each strain), were grown into single colonies, which were used to inoculate multiple independent cultures for fluctuation tests. For each culture, a single colony was resuspended in 5 ml of M9 glycerol liquid medium and grown to saturation (2 days). Tester cells were washed and resuspended in M9 buffer. Lac+ controls were diluted in M9 buffer to 1,000 cells/ml. Both testers and controls were mixed with concentrated (25x) FC29 "scavenger" cells (3) (not Lac revertible) in top agar and plated on M9 lactose medium; scavengers prevent the growth of frameshift-bearing cells on lactose selection plates by scavenging any nonlactose carbon sources. The plates were incubated at 37°C. Newly visible Lac+ colonies were scored every 2 hours, and control plates with a known dilution of Lac+ control strains were used to calculate the time at which 50% of the colonies were visible (T50) for each genotype. To avoid the risk of counting adaptive Lac+ mutants (formed after selection) (3, 28), mutants were counted at the T50 of their respective Lac+ parental strains (29), and the counts were extrapolated to estimate the number at T100. (If T50 was missed, time points between T40 and T66 were used.) The extrapolated numbers at T100 were used to determine the numbers of generation-dependent (preplating) Lac+ mutant cells per culture. Total CFU were assayed on LBH plates. Mutation rates were estimated from these mutant frequencies by the MMS maximum likelihood method (36).
Chromosomal Tet frameshift-reversion assay. Strains carrying a +1 frameshift allele of the Tn10 tetA gene disrupting the chromosomal upp gene (2) were monitored for reversion to tetracycline resistance (Tetr) in 11- to 19-tube fluctuation tests similar to those described for Lac (above) but without scavenger cells. Cells were plated on LBH with and without tetracycline and incubated at 37°C, Tetr and total colonies were scored at 48 h, and mutation rates were estimated as described above.
Measurement of SOS induction. Exonuclease-deficient and isogenic Exo+ cells carrying a chromosomal gfp reporter under the control of the sulA promoter (15, 25) were grown to saturation in M9 glycerol, concentrated, and photographed under the microscope (15) using fluorescence detection (for green fluorescent protein [GFP]) and bright field for the same fields of view. At least 12 fields were photographed per strain each day for quantification of highly GFP-positive cells.
RESULTS AND DISCUSSION
Increased spontaneous frameshift mutation rate in ExoI– ExoVII– cells at chromosomal and episomal sites. To study the frameshift mutator phenotype of exonuclease-deficient strains (46), we constructed ExoI– ExoVII– (xonA xseA) derivatives of two different +1 frameshift reporter strains for mutation rate determinations. The first carries the lacI33lacZ fusion allele on an F' episome (3) in which reversion to Lac+ can occur by a net –1 frameshift mutation within a 128-basepair region (10, 38). The second carries a chromosomal +1 frameshift allele of the tetA gene (upp::Tn10dtet+1 [2]), which confers tetracycline resistance (Tetr) upon –1 deletion at a run of five Gs (2, 9). The mutation rates in single-strand-exonuclease-proficient (Exo+) cells were 9.3 (±2) x 10–10 and 2.6 (±0.2) x 10–9 mutations per cell per generation for the episomal lac and chromosomal tet alleles, respectively (Tables 2 and 3).
We found that deletion of xonA (encoding ExoI) and xseA (encoding the large subunit of ExoVII) increased the spontaneous frameshift-mutation rates significantly: an average of 10-fold (±2-fold) for the episomal-lac reporter and 20-fold (±3-fold) for the chromosomal tet reporter when rates were compared within each experiment (Tables 2 and 3 and Fig. 1A). The data imply that an impaired ability to degrade ssDNA results in increased frameshift mutation at both chromosomal and episomal loci. Previous identification of the frameshift-mutator phenotype of cells lacking ExoI and ExoVII used assays of mutation at episomal sites (46). Those findings are confirmed and generalized here.
SOS induction in ExoI– ExoVII– cells. We used a gfp reporter gene under the control of the LexA-repressed sulA promoter to quantify GFP-positive cells as a function of SOS/LexA-regulon derepression, using fluorescence microscopy (15, 25). A previous study using this system to assay SOS induction found that mutations that lead to SOS induction can do so either in all cells or by increasing the size of a subpopulation of spontaneously green cells, or both, and both are dependent on RecA (25). The ExoI– ExoVII– strain showed increases in both kinds of induction. First, most cells appeared slightly greener than those of the isogenic Exo+ strain (Fig. 1B to E), and second, the subpopulation of very green cells was larger; the very green cells were 4.2% (81/1,922) of all cells in the ExoI– ExoVII– strain and 1.1% (22/1,937) of the isogenic Exo+ strain (data collected on two different days; P < 0.001 by z test [SigmaStat 3.1; Systat Software, Inc., Point Richmond, CA]). The greenness is RecA dependent, and thus, genuine SOS induction (25; J. M. Pennington and S. M. Rosenberg, unpublished data; and data not shown). Thus, SOS is induced in ExoI– ExoVII– cells. These data imply a role for these exonucleases in preventing/reducing spontaneous SOS induction by removal of excess ssDNA. The ssDNA normally removed by ExoI and ExoVII appears to arise at low levels in most cells, perhaps from normal DNA metabolic processes, and at higher levels/more persistently in rare cells, perhaps from sporadic spontaneous DNA damage.
Hypermutation in ExoI– ExoVII– cells requires the SOS response. The SOS DNA damage response is activated when RecA binds ssDNA, forming a nucleoprotein filament in which RecA is activated (called RecA) for facilitating autoproteolytic cleavage of target SOS response proteins, including the LexA transcriptional repressor (11). Thus, induction of SOS/LexA-controlled genes occurs neither in cells that lack RecA nor in cells that carry a special mutant allele of lexA, lexA(Ind–), that encodes an uncleavable repressor (23, 32). We observed that the increased Lac+ and Tetr frameshift mutation rates seen in ExoI– ExoVII– cells were greatly reduced in strains that also lack RecA or carry the lexA(Ind–) allele (Tables 2 and 3 and Fig. 1A). ExoI– ExoVII– RecA– cells had five- to sevenfold-lower mutation rates than isogenic ExoI– ExoVII– RecA+ controls (Fig. 1A). Similarly, the presence of the lexA(Ind–) allele decreased mutation rates three- to fourfold relative to isogenic LexA+ controls (Fig. 1A). The ExoI– ExoVII– RecA– and ExoI– ExoVII– LexA(Ind–) strains show mutation rates only slightly, and not significantly, greater than those of their respective Exo+ parent strains (Tables 2 and 3 and Fig. 1A), indicating that most of the hypermutation in ExoI– ExoVII– cells requires SOS induction.
Hypermutation in ExoI– ExoVII– cells requires DNA polymerase IV. We found that increased frameshift mutation in ExoI– ExoVII– cells required the SOS-upregulated DinB/PolIV DNA polymerase (Tables 2 and 3 and Fig. 1A). We used ExoI– ExoVII– cells that carry the dinB10 allele, which encodes a catalytically inactive enzyme (47) but is not polar on downstream genes in the dinB operon (29). dinB10 reduced the frameshift mutation rates to levels near those in Exo+ cells. Three- to 22-fold decreases relative to the isogenic ExoI– ExoVII– strains were seen, depending on the assay (Fig. 1A). In both frameshift reversion assays, the ExoI– ExoVII– PolIV– mutation rates were not significantly different from those of the respective Exo+ parent strains (Tables 2 and 3). Thus, hyper-frameshift mutation in ExoI– ExoVII– cells requires a catalytically active PolIV, implicating PolIV as the SOS-controlled component required.
Further discussion. The data reported show that E. coli strains deficient for ExoI and ExoVII single-strand-dependent exonucleases exhibit increased spontaneous frameshift mutation rates at F' episomal and chromosomal sites, as shown previously for an episomal locus (46). This increase requires RecA, a cleavable LexA/SOS repressor, and DNA PolIV (Tables 2 and 3 and Fig. 1A). The absence of ExoI and ExoVII does not diminish DNA mismatch repair activity (14, 44, 46). However, DNA metabolism is altered sufficiently to provoke SOS induction (Fig. 1B to E and see above). We note that previous analyses of the same Lac and Tet mutation assays in the Exo+ genetic background showed no PolIV-dependent (27, 29) or RecA-dependent (2, 13) spontaneous mutation; it is specific to the ExoI- ExoVII-defective background, implying that the enhanced SOS induction in ExoI- ExoVII-defective cells is responsible.
In Fig. 2, we suggest that excess linear ssDNAs are normally removed by ExoI and ExoVII, and their absence leads to SOS induction, PolIV up-regulation, and PolIV-dependent mutagenesis. PolIV might then more often displace the replicative DNA PolIII either during acts of normal DNA replication or during acts of replication in repair of spontaneous DNA double-strand breaks (34). The ssDNA ends normally removed by ExoI and ExoVII might arise during normal DNA metabolic processes, such as replication or repair, during sporadic DNA damage and repair events, or both.
The increase in the spontaneous frameshift mutation rate due to disruption of ExoI and ExoVII is less pronounced in a MG1655 or JC11450 background (data not shown) than with FC40, used here, which carries F'128. This is likely to be due to the lower level of dinB expression in those backgrounds, which lack the higher-expression episomal copy of dinB in F'128 (20). Because the initial description of increased episomal frameshift mutation in the absence of ExoI and ExoVII was also done with F'128 (46), our data are relevant to those data. Whereas the mechanism of frameshift mutation suppression by ExoI and ExoVII in that assay might have been solely via editing of frameshift mutation intermediates (46), our data indicate that an additional mechanism of suppression, by suppressing SOS induction, is also important, and in the F'128 background it appears to dominate. It is possible that some of the increase in mutation, even in this genetic background, may occur via lack of direct editing of frameshift intermediates, as proposed previously (46), because although the recA, lexA(Ind–), and dinB derivatives do not show significant differences from the Exo+ strain, they might have more mutations than Exo+ (Tables 2 and 3 and Fig. 1A).
Another low-fidelity DNA polymerase, DNA PolV, is also upregulated during SOS but is unlikely to be relevant to spontaneous mutation in ExoI– ExoVII– cells because PolV provokes substitution mutations preferentially (41), and these are not increased appreciably (14, 46). PolV becomes active only late in severe SOS responses (41). We observed only low-level SOS induction in most ExoI– ExoVII– cells (Fig. 1B to E), which may be insufficient for PolV induction, although it is mutagenic via PolIV.
Although fully redundant for mismatch repair activity (14, 44, 46), the four single-strand-specific exonucleases appear to be only partially redundant for maintenance of genomic stability. First, defects in recombination are seen when two or three are defective (30, 35, 46). Second, we show here that lack of even two can be mutagenic via increased SOS induction and PolIV up-regulation. The mutation mechanism at least partially resembles starvation stress-induced adaptive mutation in the E. coli Lac system (17) and antibiotic-induced resistance mutation (5) in that cells under starvation or antibiotic stress have increased mutation rates, depending on LexA-regulon derepression and PolIV (2, 5, 8, 26, 27). Previously, PolIV-dependent mutagenesis of E. coli was observed exclusively under conditions of starvation stress (2, 8, 27) or antibiotic stress (5) or during artificial PolIV overproduction (19, 48) and not spontaneously in growing cells (29, 49), unless the starvation stress response was artificially expressed (34). Here, the stress that promotes mutation is genomic stress, apparently from insufficient cleansing of SOS-provoking linear ssDNAs. The single-strand-specific exonucleases thus function as guardians of the genome, suppressing up-regulation of SOS genes and consequent mutagenesis.
ACKNOWLEDGMENTS
We thank Daniel B. Magner for assistance with computational aspects of data analysis; Mellanie P. Ray for technical assistance; Robert Do, Gbenga Olanrewaju, and Kenny Tran for preparation of media; and Matthew D. Blankschien, Janet L. Gibson, P. J. Hastings, Christophe Herman, Mary-Jane Lombardo, and Jeanine M. Pennington for helpful discussions and comments on the manuscript.
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