Influence of a uvrD mutation on survival and repair of X-irradiated Escherichia coli K-12 cells.

The presence of a uvrD mutation increased the X-ray sensitivities of E. coli wild-type and polA strains, but had no effect on the sensitivities of recA and recB strains, and little effect on a lexA strain. Incubation of irradiated cells in medium containing 2,4-dinitrophenol or chloramphenicol decreased the survival of wild-type and uvrD cells, but had no effect on the survival of recA, recB and lexA strains. Alkaline sucrose gradient sedimentation studies indicated that the uvrD strain is deficient in the growth-medium-dependent (Type III) repair of DNA single-strand breaks. These results indicate that the uvrD mutation inhibits certain rec+lex+-dependent repair processes, including the growth-medium-dependent (Type III) repair of X-ray-induced DNA single-strand breaks, but does not inhibit other rec+lex+-dependent processes that are sensitive to 2,4-dinitrophenol and chloramphenicol.     

Long repair replication patches are produced by the short-patch pathway in a uvrD252 (recL152) mutant of Escherichia coli K-12

The uvrD252 mutation leads to increased UV sensitivity, diminished dimer excision and host cell reactivation capacity, and an increase in the average patch size after repair replication. A recA56 uvrD252 double mutant was far more resistant to UV than was a recA56 uvrB5 double mutant. Its host cell reactivation capacity was identical to that of uvrD252 single mutant and was far greater than that of the uvrB5 single mutant. The strain showed no Weigle reactivation. From these results, we concluded that the double mutant has no inducible DNA repair (including long-patch excision repair) but retains dimer excision capabilities comparable to the uvrD252 single mutant. It appears, therefore, that the long patches detected in the uvrD mutant were not identical to the recA-dependent patches seen in wild-type cells.     

Mutation induction by monochromatic 254-nm and 365-nm radiation in strains of Escherichia coli that differ in repair capability

Mutation to tryptophan independence after exposure to radiation at the monocrhomatic wavelengths of 254 and 365 nm was studied and compared in 7 strains of Escherichia coli B/r that differ in repair capability. Efficient mutation induction was obtained with both 254-nm and 365-nm radiation with strains WP2 (wild-type), WP2s (uvrA), WP6s (polA uvrA). Mutants were not induced at either wavelength in the lexA strain WP5 or the recA strains WP10 and WP100. These results support the induction of mutants with 365-nm radiation through the error-prone (SOS) pathway of postreplication repair. Log-log plots of tryptophan revertant data at 254 nm showed the expected slopes of approximately 2.0 over the entire influence range tested. In contrast, similar plots of revertant data at 365 nm were complex in all cases tested: at low fluence values (survival greater than 0.5) in all cases where reversion occurred the slopes were approximately 1.0, while at higher fluences (survival less than 0.5) the slopes of the log-log plots were approximately 3.0 with strains WP2s and WP6s, approximately 4.0 with strain WP6 and approximately 6.0 with strain WP2. Differential sensitivity of components of excision and postreplication repair systems to 365-nm radiation may account for the 2-part mutation curves obtained with uvr⁺ rec⁺ lex⁺ strains. It is proposed that efficient error-free repair of mutational lesions occurs at 365-nm fluences below 2–4×10⁵ J m²⁻; at greater 365-nm fluences, error-free excision repair may be selectively inhibited, forcing a greater fraction of mutational lesions to be processed by the error-prone component of the postreplication repair system. The similarity of the mutational responses of WP2s and WP6 at 365 nm supports the selective inhibition of error-free excision repair.     

Current understanding of UV-induced base pair substitution mutation in E. coli with particular reference to the DNA polymerase III complex

UV mutagenesis in E. coli is believed to occur in two discrete steps. The second step involves continued DNA synthesis beyond a blocking lesion in the template strand. This bypass step requires induced levels of umuD and umuC gene products and activated recA protein. DNA polymerase III may be involved since a dnaE mutator strain (believed to have defective base selection) is associated with enhanced UV mutagenesis in conjunction with a genetic background permitting the bypass step. In non-UV-mutable umu and lexA strains, UV mutagenesis can be demonstrated if delayed photorevesal is given. This is interpreted as indicating that an earlier misincorporation step can occur in such strains but the resulting mutations do not survive because the bypass step is blocked. The misincorporation step does not require any induced SOS gene products and can occur either at the replication fork or during repair replication following excision of a DNA lesion. Neither a dnaE mutator gene (leading to a defective subunit of DNA polymerase III holoenzyme) nor a mutD5 mutator gene (leading to a defective ε proofreading subunit) had any effect on he misincorporation step. Although this is consistent with DNA polymerase III holoenzyme not being involved in the misincorporation step, other interpretations involving the inhibition of ε proofreading activity by recA protein are possible.

In vitro studies are reported in which sites of termination of synthesis by DNA polymerase III holoenzyme on UV-irradiated M13 mp8 DNA were examined in the presence of inhibitors of the 3′–5′ proofreading exonuclease (including recA protein). No evidence was found for incorporation of bases opposite photoproducts suggesting that either inhibition is more complete in the cell and/or that other factors are involved in the misincorporation step.     

Repair resynthesis in Escherichia coli mutants deficient in single-stranded DNA-binding protein

A series of Escherichia coli strains deficient in single-stranded DNA-binding protein (SSB) and DNA polymerase I was constructed in order to analyze the effects of these mutations on DNA repair resynthesis after UV-irradiation. Since SSB has been suggested to play a role in protecting single-stranded regions which may transiently exist during excision repair and since long single-stranded regions are believed to occur frequently as repair intermediates in strains deficient in DNA polymerase I, studies of repair resynthesis and strand rejoining were performed on strains containing both the ssb-1 and polA1 mutations. Repair resynthesis appears to be slightly decreased in the ssb-1 strain at 42 degrees C relative to the wild-type; however, this effect is not enhanced in a polA1 derivative of this strain. After UV-irradiation, the single-strand molecular weight of the DNA of an ssb-1 strain decreases and fails to recover to normal size. These results are discussed in the context of long patch repair as an inducible component of repair resynthesis and of the protection of intermediates in the excision repair process by SSB. A direct role for SSB in repair resynthesis involving modulation of the proteins involved in this mode of DNA synthesis (particularly stimulation of DNA polymerase II) is not supported by our findings.     

Inhibition of spontaneous mutagenesis by vanillin and cinnamaldehyde in Escherichia coli: Dependence on recombinational repair

Vanillin (VAN) and cinnamaldehyde (CIN) are dietary antimutagens that effectively inhibit both induced and spontaneous mutations. We have shown previously that VAN and CIN reduced the spontaneous mutant frequency in Salmonella TA104 (hisG428, rfa, ΔuvrB, pKM101) by approximately 50% and that both compounds significantly reduced mutations at GC sites but not at AT sites. Previous studies have suggested that VAN and CIN may reduce mutations in bacterial model systems by modulating DNA repair pathways, particularly by enhancing recombinational repair. To further explore the basis for inhibition of spontaneous mutation by VAN and CIN, we have determined the effects of these compounds on survival and mutant frequency in five Escherichia coli strains derived from the wild-type strain NR9102 with different DNA repair backgrounds. At nontoxic doses, both VAN and CIN significantly reduced mutant frequency in the wild-type strain NR9102, in the nucleotide excision repair-deficient strain NR11634 (uvrB), and in the recombination-proficient but SOS-deficient strain NR11475 (recA430). In contrast, in the recombination-deficient and SOS-deficient strain NR11317 (recA56), both VAN and CIN not only failed to inhibit the spontaneous mutant frequency but actually increased the mutant frequency. In the mismatch repair-defective strain NR9319 (mutL), only CIN was antimutagenic. Our results show that the antimutagenicity of VAN and CIN against spontaneous mutation required the RecA recombination function but was independent of the SOS and nucleotide excision repair pathways. Thus, we propose the counterintuitive notion that these antimutagens actually produce a type of DNA damage that elicits recombinational repair (but not mismatch, SOS, or nucleotide excision repair), which then repairs not only the damage induced by VAN and CIN but also other DNA damage—resulting in an antimutagenic effect on spontaneous mutation.     

Specificity of N-acetoxy-N-2-acetylaminofluorene-induced frameshift mutation spectrum in mismatch repair deficient Escherichia coli strains mutH, L, S and U

The mismatch repair system of Escherichia coli is known to contribute to the fidelity of the replicational process. This system involves the functions of mutH, mutL, mutS and mutU (uvrD) loci which recognize mispaired bases as a consequence of errors due to the polymerase itself. Chemical modifications of DNA have also been suspected to create mispaired bases which, if the mispaired bases are removed, will lead to mutations by frameshift. Using the pBR322 plasmid DNA modified by the ultimate carcinogen N-acetoxy-N-2-acetylaminofluorene (N-Aco-AAF) we have investigated this possibility in a forward mutational assay (tetracycline sensitivity). This fluorene derivative has been shown to induce predominantly frameshift mutations. Our results show that: The sensitivity of the deficient strains mutH, mutL and mutS to the AAF adducts is similar to that of the corresponding wild-type strain. However, the mutU strain appears much more sensitive to those adducts although less than a uvrA, B or C-deficient strain. This suggests that the mutU gene product is involved in the repair of AAF adducts. For the four mut deficient strains, and as it was shown with the wild-type strain, AAF adducts induced mutations to tetracycline sensitivity are only observed when the SOS system of the host bacteria is induced by irradiation of the cells prior to transformation with the modified plasmid. The mutation frequencies depend upon the ultraviolet light doses and similar maxima were found for the four mut strains and the corresponding wild-type strain. In agreement with the results obtained with wild-type or uvrA strains we observe that AAF adducts induce mostly frameshift mutations in the mut strains. Two types of hot spots of mutagenesis were described in wild-type and uvrA strains occurring either at repetitive sequences or at sequences of the type 5' G-G-C-G-C-C 3' (NarI restriction enzyme recognition sequence). While the second type of mutational hot spot does exist in the mismatch repair-deficient strains, we observe that the repetitive sequences are no longer hot spots of mutations in these strains, suggesting that the mismatch repair protein complex is involved in the establishment of AAF-induced frameshift mutations at repetitive sequences.     

The mismatch repair system (mutS, mutL and uvrD genes) in Pseudomonas aeruginosa: molecular characterization of naturally occurring mutants

We have recently described the presence of a high proportion of Pseudomonas aeruginosa isolates (20%) with an increased mutation frequency (mutators) in the lungs of cystic fibrosis (CF) patients. In four out of 11 independent P. aeruginosa strains, the high mutation frequency was found to be complemented with the wild-type mutS gene from P. aeruginosa PAO1. Here, we report the cloning and sequencing of two additional P. aeruginosa mismatch repair genes and the characterization, by complementation of deficient strains, of these two putative P. aeruginosa mismatch repair genes (mutL and uvrD). We also describe the alterations in the mutS, mutL and uvrD genes responsible for the mutator phenotype of hypermutable P. aeruginosa strains isolated from CF patients. Seven out of the 11 mutator strains were found to be defective in the MMR system (four mutS, two mutL and one uvrD). In four cases (three mutS and one mutL), the genes contained frameshift mutations. The fourth mutS strain showed a 3.3 kb insertion after the 10th nucleotide of the mutS gene, and a 54 nucleotide deletion between two eight nucleotide direct repeats. This deletion, involving domain II of MutS, was found to be the main one responsible for mutS inactivation. The second mutL strain presented a K310M mutation, equivalent to K307 in Escherichia coli MutL, a residue known to be essential for its ATPase activity. Finally, the uvrD strain had three amino acid substitutions within the conserved ATP binding site of the deduced UvrD polypeptide, showing defective mismatch repair activity. Interestingly, cells carrying this mutant allele exhibited a fully active UvrABC-mediated excision repair. The results shown here indicate that the putative P. aeruginosa mutS, mutL and uvrD genes are mutator genes and that their alteration results in a mutator phenotype.     

Repair resynthesis in Escherichia coli mutants deficients in single-stranded DNA-binding protein

A series of Escherichia coli strains deficients in single-stranded DNA-binding protein (SSB) and DNA polymerase I was constructed in order to analyze the effects of these mutations on DNA repair resynthesis after UV-irradiation. Since SSB has been suggested to play a role in protecting single-stranded regions which may transiently exist during excision repair and since long single-stranded regions are believed to occur frequently as repair intermediates in strains deficient in DNA polymerase I, studies of repair resynthesis and strand rejoining were performed on strains containing both the ssb-1 and polA1 mutations. Repair resynthesis appears to be slightly decreased in the ssb-1 strain at 42°C relative to the wild-type; however, this effect is not enhanced in a polA1 derivative of this strain. After UV-irradiation, the single-strand molecular weight of the DNA of an ssb-1 strain decreases and fails to recover to normal size. These results are discussed in the context of long patch repair as an inducible component of repair resynthesis and of the protection of intermediates in the excision repair process by SSB. A direct role for SSB in repair resynthesis involving modulation of the proteins involved in this mode of DNA synthesis (particularly stimulation of DNA polymerase II) is not supported by our findings.     

Genetic Analysis of δheld and δuvrd Mutations in Combination with Other Genes in the Recf Recombination Pathway in Escherichia Coli: Suppression of a Ruvb Mutation by a Uvrd Deletion

Helicase II (uvrD gene product) and helicase IV (helD gene product) have been shown previously to be involved in the RecF pathway of recombination. To better understand the role of these two proteins in homologous recombination in the RecF pathway [recBCsbcB(C) background], we investigated the interactions between helD, uvrD and the following RecF pathway genes: recF, recO, recN and ruvAB. We observed synergistic interactions between uvrD and the recF, recN, recO and recG genes in both conjugational recombination and the repair of methylmethane sulfonate (MMS)-induced DNA damage. No synergistic interactions were detected between helD and the recF, recO and recN genes when conjugational recombination was analyzed. We did, however, detect synergistic interactions between helD and recF/recO in recombinational repair. Suprisingly, the uvrD deletion completely suppressed the phenotype of a ruvB mutation in a recBCsbcB(C) background. Both conjugational recombination efficiency and MMS-damaged DNA repair proficiency returned to wild-type levels in the δuvrDruvB9 double mutant. Suppression of the effects of the ruvB mutation by a uvrD deletion was dependent on the recG and recN genes and not dependent on the recF/O/R genes. These data are discussed in the context of two ``RecF' homologous recombination pathways operating in a recBCsbcB(C) strain background.     

lon incompatibility associated with mutations causing SOS induction: null uvrD alleles induce an SOS response in Escherichia coli

The uvrD gene in Escherichia coli encodes a 720-amino-acid 3'-5' DNA helicase which, although nonessential for viability, is required for methyl-directed mismatch repair and nucleotide excision repair and furthermore is believed to participate in recombination and DNA replication. We have shown in this study that null mutations in uvrD are incompatible with lon, the incompatibility being a consequence of the chronic induction of SOS in uvrD strains and the resultant accumulation of the cell septation inhibitor SulA (which is a normal target for degradation by Lon protease). uvrD-lon incompatibility was suppressed by sulA, lexA3(Ind(-)), or recA (Def) mutations. Other mutations, such as priA, dam, polA, and dnaQ (mutD) mutations, which lead to persistent SOS induction, were also lon incompatible. SOS induction was not observed in uvrC and mutH (or mutS) mutants defective, respectively, in excision repair and mismatch repair. Nor was uvrD-mediated SOS induction abolished by mutations in genes that affect mismatch repair (mutH), excision repair (uvrC), or recombination (recB and recF). These data suggest that SOS induction in uvrD mutants is not a consequence of defects in these three pathways. We propose that the UvrD helicase participates in DNA replication to unwind secondary structures on the lagging strand immediately behind the progressing replication fork, and that it is the absence of this function which contributes to SOS induction in uvrD strains.     

Effect of the uvrD mutation on excision repair.

A pair of related Escherichia coli K-12 strains, one of which contains the uvrD101 mutation, were constructed and compared for ability to perform various steps in the excision repair of deoxyribonucleic acid damage inflicted by ultraviolet radiation. The results of this study indicated: (i) ultraviolet sensitivity in the uvrD101 mutant was greater than that of wild type but less than that measured in an incision-deficient uvrA mutant; (ii) host cell reactivation paralleled the survival data; (iii) postirradiation deoxyribonucleic acid degradation was virtually identical in the two strains; (iv) incision, presumably at the sites of pyrimidine dimers, proceeded normally in the uvrD101 strain; (v) excision of pyrimidine dimers was markedly reduced in both rate and extent in the uvrD101 mutant; (vi) the amount of repair resynthesis was the same in both strains, and there was no evidence of abnormally long repair patches in the uvrD mutant; and (vii) rejoining of incision breaks was slow and incomplete in the uvrD strain. These data suggest that the ultraviolet sensitivity conferred by the uvrD mutation arises from inefficient removal of pyrimidine dimers or from failure to close incision breaks. The data are compatible with the notion that the uvrD+ gene produce affects the conformation of incised deoxyribonucleic acid molecules.     

Mutations in uvrD induce the SOS response in Escherichia coli.

We have isolated three new mutations in uvrD that increase expression of the Escherichia coli SOS response in the absence of DNA damage. Like other uvrD (DNA helicase II) mutants, these strains are sensitive to UV irradiation and have high spontaneous mutation frequencies. Complementation studies with uvrD+ showed that UV sensitivity and spontaneous mutator activity were recessive in these new mutants. The SOS-induction phenotype, however, was not completely complemented, which indicated that the mutant proteins were functioning in some capacity. The viability of one of the mutants in combination with rep-5 suggests that the protein is functional in DNA replication. We suggest that these mutant proteins are deficient in DNA repair activities (since UV sensitivity is complemented) but are able to participate in DNA replication. We believe that defective DNA replication in these mutants increases SOS expression.     

Gene conversion in Escherichia coli. Resolution of heteroallelic mismatched nucleotides by co-repair

We have constructed heteroduplex plasmid DNA that is similar in structure to the heteroduplex DNA expected to be produced during genetic recombination of plasmids, and studied its repair after transformation into different Escherichia coli strains. The heteroduplex DNA was constructed using two different parental plasmids, each of which contained a different ten-nucleotide insertion mutation. The effect of different defined states of dam-methylation on repair was also examined. We found that heteroduplex DNA repair occurred prior to the replication of the substrate DNA 60 to 80% of the time, regardless of the state of DNA methylation. Most excision/synthesis tracts covered two markers separated by 1243 base-pairs, and this process has been termed co-repair. The most efficient co-repair pathway was the Dam-instructed repair pathway that required the mutH, mutL, mutS and uvrD gene products and preferentially used the methylated strand as the template for DNA synthesis. If there was no methylation asymmetry, mismatch nucleotide repair occurred with a similar frequency; however, no strand bias was observed. Co-repair of symmetrically methylated heteroduplex DNA required the mutS and uvrD gene products, while repair of unmethylated heteroduplex DNA also required the mutL and mutH gene products.     

The rad2 mutation affects the molecular nature of UV and acridine-mustard-induced mutations in the ADE2 gene of Saccharomyces cerevisiae

We have studied the molecular nature of ade2 mutations induced by UV light and bifunctional acridine-mustard (BAM) in wild-type (RAD) and in excision-deficient (rad2) strains of the yeast, Saccharomyces cerevisiae. In the RAD strain, UV causes 45% GC → AT transitions among all mutations; in the rad2 strain this value is 77%. BAM was shown to be highly specific for frameshift mutagenesis: 60% frameshifts in the RAD strain, and as many as 84% frameshifts in the rad2 strain were induced. Therefore, the rad2 mutation affects the specificity of UV- and BAM-induced mutagenesis in yeast. Experimental data agree with the view that the majority of mutations in the RAD strain are induced by a prereplicative mechanism, whereas mutations in the RAD strain are induced by a prereplicative mechanism, whereas mutations in the rad2 strain are predominantly postreplicative events. Our results also suggest that: (1) cytosine-containing photoproducts are the substances responsible for major premutational damage to DNA; (2) a fraction of the mutations may arise in the course of excision repair of UV photoproducts.     

Involvement of recQ in the ultraviolet damage repair pathway in Deinococcus radiodurans

Deinococcus radiodurans is a bacterium which can survive extremely DNA damage. To investigate the relationship between recQ and the ultraviolet radiation (UV) damage repair pathway, we created a four mutant strain by constructing recQ knockout mutants in uvrA1, uvrA2, and uvsE backgrounds. Using the rpoB/Rif^r system, we measured the mutation frequencies and rates in wild type, recQ (MQ), uvsE uvrA1 uvrA2 (TNK006), and uvsE uvrA1 uvrA2 recQ (TQ). We then isolated Rif^r mutants of these strains and sequenced the rpoB gene. The mutation frequency of TQ was 6.4, 10.1, and 2.43 times that of wild type, MQ, and TNK006, respectively, and resulted in rates of 4.7, 6.71, and 2.15 folds higher than that of wild type, MQ, and TNK006, respectively. All the strains demonstrated specific mutational hotspots. Furthermore, the TQ strain showed a transversion bias that was different from the other three strains. The results indicate that recQ is involved in the ultraviolet damage repair pathway via the interaction between recQ and uvrA1, uvrA2, and uvsE in D. radiodurans.     

Characterization of MMS-sensitive mutants of Neurospora crassa

Several MMS-sensitive mutants of Neurospora crassa were compared with the wild-type strain for their relative sensitivities to UV, X-ray, and histidine. They were also compared for the frequency of spontaneous mutation at the loci which confer resistance to p-fluorophenylalanine. The mutants were also examined for possible defects in meiotic behavior in homozygous crosses and for any change in the inducible DNA salvage pathways (as indicated by their ability to utilize DNA as the sole phosphate source in the growth medium). On the basis of these characterizations, the present MMS-sensitive mutants of Neurospora can be placed into three groups. The first group includes three mutants, mus-(SC3), mus-(SC13), and mus-(SC28). These are slow growers, insensitive to histidine with no apparent meiotic defects and may have reduced frequency of spontaneous mutation. In addition, their mycelial growth is sensitive to MMS but the conidial viability following MMS, UV or X-ray treatment appears normal or only slightly more sensitive than the wild-type. The second group includes only one mutant, mus-(SC15); its mycelial growth is very sensitive to MMS but the conidial survival following treatment with MMS or UV appears normal; however, the conidial survival following exposure to X-ray is significantly reduced. This mutant shows an increase (more than 10-fold) frequency of spontaneous mutation, but behaves normal like the wild-type with respect to fertility, growth rate and insensitivity to histidine. The third group includes mutants mus-(SC10), mus-(SC25), and mus-(SC29). These mutants are very sensitive to UV, X-rays and MMS and to histadine but have normal growth rates on minimal medium. Mutant mus-(SC10), but not mus-(SC25) and mus-(SC29), has an increased (11 ×) frequency of spontaneous mutation. On the basis of data presented, the MMS sensitivity of the first group of mutants cannot be ascertained to arise from a defect in the DNA repair pathways; instead, it may stem from altered cell permeability or other pleotropic effects of the mus mutations. However, it can be suggested that the second and third group of mus mutants may indeed result from a defect in the DNA repair pathways controlled by the mus genes; this conclusion is based on their cross-sensitivity to a number of DNA-damaging agents such as MMS, UV and/or X-ray, high frequencies of spontaneous mutation (mutator effects) and defects in meiotic behavior.     

The influence of deficiencies in DNA-repair on MR-mediated reversion of an insertion-sequence mutation in Drosophila melanogaster

MR is a frequently occurring mutator in Drosophila melanogaster inducing mutation by the incorporation of insertion sequences. In the presence of MR a mutation at the singed (sn) locus induced by MR, reverts to wild-type at a high frequency of 1.7%. This reversion system which presumably involves the removal of an insertion element, was used to study the effects of defective DNA repair. Thus, reversion frequencies were compared in progeny of flies with mei-9, deficient for excision repair, mei-41, deficient for post-replication repair, or with both mei-9 and mei-41. The data show that under conditions of defective DNA repair, the frequency of MR-mediated reversion, is consistently decreased in comparison to repair-proficient conditions. This effect is explained by assuming that defective repair interferes with some steps in the process of reverse mutation involving the removal of insertion sequences. The observed reduction in reversion frequency may well result from selective elimination of cells in which the reversion process has not been completed.     

Spontaneously arising mutL mutators in evolving Escherichia coli populations are the result of changes in repeat length

Over the course of thousands of generations of growth in a glucose-limited environment, 3 of 12 experimental populations of Escherichia coli spontaneously and independently evolved greatly increased mutation rates. In two of the populations, the mutations responsible for this increased mutation rate lie in the same region of the mismatch repair gene mutL. In this region, a 6-bp repeat is present in three copies in the gene of the wild-type ancestor of the experimental populations but is present in four copies in one of the experimental populations and two copies in the other. These in-frame mutations either add or delete the amino acid sequence LA in the MutL protein. We determined that the replacement of the wild-type sequence with either of these mutations was sufficient to increase the mutation rate of the wild-type strain to a level comparable to that of the mutator strains. Complementation of strains bearing the mutator mutations with wild-type copies of either mutL or the mismatch repair gene uvrD rescued the wild-type mutation rate. The position of the mutator mutations-in the region of MutL known as the ATP lid-suggests a possible deficiency in MutL's ATPase activity as the cause of the mutator phenotype. The similarity of the two mutator mutations (despite the independent evolutionary histories of the populations that gave rise to them) leads to a discussion of the potential adaptive role of DNA repeats.     

uvrD mutations enhance tandem repeat deletion in the Escherichia coli chromosome via SOS induction of the RecF recombination pathway

It has previously been shown that recombination between tandem repeats is not significantly affected by a recA mutation in Escherichia coli . Here, we describe the activation of a RecA-dependent recombination pathway in a hyper-recombination mutant. In order to analyse how tandem repeat deletion may proceed, we searched for mutants that affect this process. Three hyper-recombination clones were characterized and shown to be mutated in the uvrD gene. Two of the mutations were identified as opal mutations at codons 130 and 438. A uvrD  ::Tn 5 mutation was used to investigate the mechanism of deletion formation in these mutants. The uvrD -mediated stimulation of deletion was abolished by a lexAind3 mutation or by inactivation of either the recA , recF , recQ or ruvA genes. We conclude that (i) this stimulation requires SOS induction and (ii) tandem repeat recombination in uvrD mutants occurs via the RecF pathway. In uvrD ⁺ cells, constitutive expression of SOS genes is not sufficient to stimulate deletion formation. This suggests that the RecF recombination pathway activated by SOS induction is antagonized by the UvrD protein. Paradoxically, we observed that the overproduction of UvrD from a plasmid also stimulates tandem repeat deletion. However, this stimulation is RecA independent, as is deletion in a wild-type strain. We propose that the presence of an excess of the UvrD helicase favours replication slippage. This work suggests that the UvrD helicase controls a balance between different routes of tandem repeat deletion.