Isolation and properties of a recombination-deficient mutant of Micrococcus radiodurans.

A mutant of micrococcus radiodurans which is deficient in recombination has been isolated after treatment of the wild type with N-methyl-N'-nitro-N-nitrosoguanidine. We have called this mutant Micrococcus radiodurans rec30. The efficiency of recombination in this mutant, as measured by transformation, is less than 0.01% that of the wild type. It is 15 times more sensitive to the lethal action of ultraviolet radiation, 120 times more sensitive to ionizing radiation, and 300 times more sensitive to mitomycin C (MMC) than the wild type. It is probably inactivated by a single MMC-induced deoxyribonucleic acid cross-link per genome. The excision of ultraviolet-induced pyrimidine dimers is normal. There is no radiation-induced degradation of deoxyribonucleic acid. All spontaneous revertants selected for resistance to low levels of MMC had wild-type resistance to radiation and MMC, and the same efficiency of recombination as the wild type, suggesting that the recombination deficiency of the strain is due to a single mutation. Deoxyribonucleic acid from this mutant can transform M. radiodurans UV17 presumed deficient in an exr type gene to wild type.     

The DNA excision repair system of the highly radioresistant bacterium Deinococcus radiodurans is facilitated by the pentose phosphate pathway

Deinococcus radiodurans is highly resistant to radiation and mutagenic chemicals. Mutants defective in the putative glucose-6-phosphate dehydrogenase gene (zwf-) and the aldolase gene (fda-) were generated by homologous recombination. These mutants were used to test the cells' resistance to agents that cause dimer formation and DNA strand breaks. The zwf - mutants were more sensitive to agents that induce DNA excision repair, such as UV irradiation and H2O2, but were as resistant to DNA strand break-causing agents such as methylmethanesulphonic acid (MMS) and mitomycin C (MMC) as the wild-type cells. Analysis of the cytoplasmic fraction of zwf- cells showed that the concentrations of inosine monophosphate (IMP) and uridine monophosphate (UMP) were only 30% of those found in the wild-type cells. The fda- mutants were slightly more resistant to UV light and H2O2. Results suggested that the deinococcal pentose phosphate pathway augmented the DNA excision repair system by providing cells with adequate metabolites for the DNA mismatch repair.     

Interspecific recombination between Escherichia coli and Salmonella typhimurium occurs by the RecABCD pathway.

Interspecific recombination in conjugation between Escherichia coli and Salmonella typhimurium is several orders of magnitude lower than intraspecies recombination and is dependent on the RecA function. This low efficiency is due to a 20% divergence in the DNA sequence. The methyl-directed (mut H,L,S dependent) mismatch repair system appears to control the fidelity of homologous recombination; inactivating one of the Mut functions increases the interspecies recombination at least by 10(3)-fold. The interspecific recombination in mutS or mutL mutants is only approximately 10-fold lower than recombination in homospecific crosses as found after correction for the efficiency of mating and DNA transfer by zygotic induction experiments. The interspecific recombination is dependent on the RecABCD pathway: it was abolished in a recA mutant and decreased approximately 10(3)-fold in a recC mutant.     

Effect of mutations in genes affecting homologous recombination on restriction enzyme-mediated and illegitimate recombination in Saccharomyces cerevisiae.

Restriction enzyme-mediated events (REM events; integration of transforming DNA catalyzed by in vivo action of a restriction enzyme) and illegitimate recombination events (IR events; integration of transforming DNA that shares no homology with the host genomic sequences) have been previously characterized in Saccharomyces cerevisiae. This study determines the effect of mutations in genes that are involved in homologous recombination and/or in the repair of double-stranded DNA breaks on these recombination events. Surprisingly, REM events are completely independent of the double-strand-break repair functions encoded by the RAD51, RAD52, and RAD57 genes but require the RAD50 gene product. IR events are under different genetic control than homologous integration events. In the rad50 mutant, homologous integration occurred at wild-type frequency, whereas the frequency of IR events was 20- to 100-fold reduced. Conversely, the rad52 mutant was grossly deficient in homologous integration (at least 1,000-fold reduced) but showed only a 2- to 8-fold reduction in IR frequency.     

Defective Excision Repair of Pyrimidine Dimers in the Ultraviolet-Sensitive Escherichia coli ras− Mutant

The ras(-) mutant of Escherichia coli K-12 is sensitive to ultraviolet (UV) light but only slightly sensitive to X-irradiation (1.5-fold increase). Other phenotypic properties include normal recombination ability and normal host cell reactivation ability but an abnormally high frequency of UV-induced mutation. The response of the ras(-) mutant to UV has been studied biochemically. After low doses of UV, the ras(-) mutant degraded excessive amounts of deoxyribonucleic acid, and long delays in resumption of deoxyribonucleic acid synthesis occurred. Pyrimidine dimers were excised at the normal rate. Although the mutant had the capability of initiating repair replication, the process was not completed after the high UV dose required to allow detection of repair replication. The ras(-) mutant, after low UV doses, left three to four times as many single-strand breaks not rejoined as did the wild-type strain.     

The role of Deinococcus radiodurans RecFOR proteins in homologous recombination

Deinococcus radiodurans exhibits extraordinary resistance to the lethal effect of DNA-damaging agents, a characteristic attributed to its highly proficient DNA repair capacity. Although the D. radiodurans genome is clearly devoid of recBC and addAB counterparts as RecA mediators, the genome possesses all genes associated with the RecFOR pathway. In an effort to gain insights into the role of D. radiodurans RecFOR proteins in homologous recombination, we generated recF, recO and recR disruptant strains and characterized the disruption effects. All the disruptant strains exhibited delayed growth relative to the wild-type, indicating that the RecF, RecO and RecR proteins play an important role in cell growth under normal growth conditions. A slight reduction in transformation efficiency was observed in the recF and recO disruptant strains compared to the wild-type strain. Interestingly, disruption of recR resulted in severe reduction of the transformation efficiency. On the other hand, the recF disruptant strain was the most sensitive phenotype to γ rays, UV irradiation and mitomycin C among the three disruptants. In the recF disruptant strain, the intracellular level of the LexA1 protein did not decrease following γ irradiation, suggesting that a large amount of the RecA protein remains inactive despite being induced. These results demonstrate that the RecF protein plays a crucial role in the homologous recombination repair process by facilitating RecA activation in D. radiodurans. Thus, the RecF and RecR proteins are involved in the RecA activation and the stability of incoming DNA, respectively, during RecA-mediated homologous recombination processes that initiated the ESDSA pathway in D. radiodurans. Possible mechanisms that involve the RecFOR complex in homologous intermolecular recombination and homologous recombination repair processes are also discussed.     

Cytogenetical characterization of UV-sensitive repair-deficient CHO cell line 43-3B. II. Induction of cell killing, chromosomal aberrations and sister-chromatid exchanges by 4NQO, mono- and bi-functional alkylating agents

An established cell line of Chinese hamster ovary (CHO-9) cells and its UV-sensitive mutant 43-3B have been studied for the induction of cell killing, chromosomal aberrations and sister-chromatid exchanges (SCEs) after exposure to different types of DNA-damaging agents such as 4-nitroquinoline-1-oxide (4NQO), mitomycin C (MMC), diepoxybutane (DEB), methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS) and ethyl nitrosourea (ENU). In comparison with the wild-type CHO cells, 43-3B cells showed very high sensitivity to the UV-mimetic agent 4NQO and the DNA cross-linking agents MMC and DEB. The 43-3B cells responded with higher sensitivity to the monofunctional alkylating agents (MMS, EMS and ENU). The increased cytotoxic effects of all these chemicals correlated well with the elevated increase in the frequency of chromosomal aberrations. In 43-3B cells exposed to 4NQO, MMC or DEB the increase in the frequency of chromosomal aberrations was much higher than the increase in the frequency of SCEs (4-10-fold) when compared to the wild-type CHO cells. This suggests that SCEs are results of fundamentally different cellular events. The responses of 43-3B cells to UV, 4NQO, MMC and DEB resemble those of 2 human syndromes, i.e., xeroderma pigmentosum and Fanconi's anemia. These data suggest that 43-3B cells are defective in excision repair as well as the other pathways involved in the repair of cross-links (MMC, DEB) and bulky DNA adducts (4NQO).     

Inactivation of mismatch repair increases the diversity of Vibrio parahaemolyticus

Inactivation of mismatch repair (MMR) has been shown to increase the accumulation of spontaneous mutations and frequency of recombination for diverse pathogenic bacteria. Currently, little is known regarding the role of mutator phenotypes for the diversification of natural populations of opportunistic human pathogens in marine environments. In this study, a higher frequency of mutators was detected among V. parahaemolyticus strains obtained from environmental sources compared with clinical sources. Inactivation of the MMR gene mutS caused increased antibiotic resistance and phase variation resulting in translucent colony morphologies. Increased nucleotide diversity in mutS and rpoB alleles from mutator compared with wild-type strains indicated a significant contribution of the mutator phenotype to the evolution of select genes. The results of this study indicate that the inactivation of MMR in V. parahaemolyticus leads to increased genetic and phenotypic diversity. This study is the first to report a higher frequency of natural mutators among Vibrio environmental strains and to provide evidence that inactivation of MMR increases the diversity of V. parahaemolyticus .     

Escherichia coli ras locus: its involvement in radiation repair

There are several classes of Escherichia coli mutants defective in radiation repair. These include strains defective in pyrimidine dimer excision, in photoreactivation, in recombination, in repair of X-ray damage, and ultraviolet (UV)-conditional mutants which do not divide after UV. Another mutant (ras(-)) has been isolated. The ras(-) has increased UV sensitivity, but only slightly increased X-ray sensitivity (1.5-fold increase). Ability to effect genetic recombination, to reactivate irradiated bacteriophage T1, and to be photoreactivated is normal. UV-induced mutation frequency is greatly increased in the mutant. The ras(-) apparently lacks the ability to repair some UV damage in the bacterial cell but can repair UV damage to bacteriophage DNA. The ras locus is located between lac and purE on the chromosome map.     

Dominant negative mutator mutations in the mutS gene of Escherichia coli.

The MutS protein of Escherichia coli is part of the dam-directed MutHLS mismatch repair pathway which rectifies replication errors and which prevents recombination between related sequences. In order to more fully understand the role of MutS in these processes, dominant negative mutS mutations on a multicopy plasmid were isolated by screening transformed wild-type cells for a mutator phenotype, using a Lac+ papillation assay. Thirty-eight hydroxylamine- and 22 N-methyl-N'-nitro-N-nitrosoguanidine-induced dominant mutations were isolated. Nine of these mutations altered the P-loop motif of the ATP-binding site, resulting in four amino acid substitutions. With one exception, the remaining sequenced mutations all caused substitution of amino acids conserved during evolution. The dominant mutations in the P-loop consensus caused severely reduced repair of heteroduplex DNA in vivo in a mutS mutant host strain. In a wild-type strain, the level of repair was decreased by the dominant mutations to between 12 to 90% of the control value, which is consistent with interference of wild-type MutS function by the mutant proteins. Increasing the wild-type mutS gene dosage resulted in a reversal of the mutator phenotype in about 60% of the mutant strains, indicating that the mutant and wild-type proteins compete. In addition, 20 mutant isolates showed phenotypic reversal by increasing the gene copies of either mutL or mutH. There was a direct correlation between the levels of recombination and mutagenesis in the mutant strains, suggesting that these phenotypes are due to the same function of MutS.     

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.     

Conjugational hyperrecombination achieved by derepressing the LexA regulon,altering the properties of RecA protein and inactivating mismatch repair in Escherichia coli K-12

The frequency of recombinational exchanges (FRE) that disrupt co-inheritance of transferred donor markers in Escherichia coli Hfr by F(-) crosses differs by up to a factor of two depending on physiological factors and culture conditions. Under standard conditions we found FRE to be 5.01 +/- 0.43 exchanges per 100-min units of DNA length for wild-type strains of the AB1157 line. Using these conditions we showed a cumulative effect of various mutations on FRE. Constitutive SOS expression by lexA gene inactivation (lexA71::Tn5) and recA gene mutation (recA730) showed, respectively, approximately 4- and 7-fold increases of FRE. The double lexA71 recA730 combination gave an approximately 17-fold increase in FRE. Addition of mutS215::Tn10, inactivating the mismatch repair system, to the double lexA recA mutant increased FRE to approximately 26-fold above wild-type FRE. Finally, we showed that another recA mutation produced as much SOS expression as recA730 but increased FRE only 3-fold. We conclude that three factors contribute to normally low FRE under standard conditions: repression of the LexA regulon, the properties of wild-type RecA protein, and a functioning MutSHL mismatch repair system. We discuss mechanisms by which the lexA, recA, and mutS mutations may elevate FRE cumulatively to obtain hyperrecombination.     

Functional analysis of Borrelia burgdorferi uvrA in DNA damage protection

Bacterial pathogens face constant challenges from DNA-damaging agents generated by host phagocytes. Although Borrelia burgdorferi appears to have much fewer DNA repair enzymes than pathogens with larger genomes, it does contain homologues of uvrA and uvrB (subunits A and B of excinuclease ABC). As a first step to exploring the physiologic function of uvrA(Bbu) and its possible role in survival in the host in the face of DNA-damaging agents, a partially deleted uvrA mutant was isolated by targeted inactivation. While growth of this mutant was markedly inhibited by UV irradiation, mitomycin C (MMC) and hydrogen peroxide at doses that lacked effect on wild-type B. burgdorferi, its response to pH 6.0-6.8 and reactive nitrogen intermediates was similar to that of the wild-type parental strain. The sensitivity of the inactivation mutant to UV irradiation, MMC and peroxide was complemented by an extrachromosomal copy of uvrA(Bbu). We conclude that uvrA(Bbu) is functional in B. burgdorferi.     

Mitotic recombination and inactivation in Saccharomyces cerevisiae induced by UV-radiation (254 nm) and hyperthermia depend on UV fluence rate

In experiments with wild-type diploid yeast cells of Saccharomyces cerevisiae, the synergistic interaction of ultraviolet (UV) light (wavelength, 254 nm) and heat (45--60 degrees C) was studied both for mutagenic and inactivation effects. Simultaneous hyperthermia and UV light treatments increase the frequency of UV-induced mitotic intergenic recombination (crossing-over) and cell inactivation. The enhancing effect was a function of UV light fluence rate. It is concluded that the effect of hyperthermia on low fluence UV or high fluence UV irradiation results in comparable effects on survival and mitotic recombination suggesting similar modulation by hyperthermia of the effects induced by UV at different fluence rates. The interpretation of the data obtained was carried out within the widely accepted point of view considering the synergistic effects as a result of repair ability damage.     

Cytogenetical characterisation of Chinese hamster 43-3B transferants with the amplified or non-amplified human DNA repair gene ERCC-1

A comparative study on the biological responses to different mutagens (UV, 4NQO, MMC, MMS and EMS) was made on CHO wild-type cells (CHO-9), its UV-hypersensitive mutant 43-3B, and 2 types of its transferants, i.e., one containing a few copies of the human repair gene ERCC-1 and the other having more than 100 copies of ERCC-1 (due to gene amplification). Cell survival, chromosomal aberrations and SCEs were used as biological end-points. The spontaneous frequency of chromosomal aberrations in the transferants was less than found in 43-3B mutant cells, but still 2-3 times higher than in wild-type CHO cells. The spontaneous frequency of SCEs in the transferants was less than in 43-3B and similar to that of wild-type cells. The induction of SCEs by all tested agents in transferants was similar to that found in CHO-9 cells, while the mutant is known to respond with higher frequencies. ERCC-1 also bestowed resistance to MMS and EMS on the mutant to induction of chromosomal aberrations and cell killing to levels comparable with those of the wild-type strain. On the other hand ERCC-1 could not completely regain the repair proficiency against cell killing and induction of chromosomal aberrations by UV or MMC to the wild-type level. These results suggest that the ERCC-1 corrects the repair defect in CHO mutant cells, but it is unable to rectify fully the defect; probable reasons for this are discussed. However, amplified transferants (having more than 100 copies of the ERCC-1 gene) restored the impaired repair function in 43-3B to UV-, MMC- or 4NQO-induced DNA damage better than non-amplified transferants with a few copies of the ERCC-1. This difference may be due to the high amount of gene product involved in the excision repair process in the amplified cells.     

Restriction enzymes increase efficiencies of illegitimate DNA integration but decrease homologous integration in mammalian cells

Mammalian cells repair DNA double-strand breaks by illegitimate end-joining or by homologous recombination. We investigated the effects of restriction enzymes on illegitimate and homologous DNA integration in mammalian cells. A plasmid containing the neo^R expression cassette, which confers G418 resistance, was used to select for illegitimate integration events in CHO wild-type and xrcc5 mutant cells. Co-transfection with the restriction enzymes BamHI, BglII, EcoRI and KpnI increased the efficiency of linearized plasmid integration up to 5-fold in CHO cells. In contrast, the restriction enzymes did not increase the integration efficiency in xrcc5 mutant cells. Effects of restriction enzymes on illegitimate and homologous integration were also studied in mouse embryonic stem (ES) cells using a plasmid containing the neo^R gene flanked by exon 3 of Hprt. The enzymes BamHI, BglII and EcoRI increased the illegitimate integration efficiency of transforming DNA several-fold, similar to the results for CHO cells. However, all three enzymes decreased the absolute frequency of homologous integration ~2-fold, and the percentage of homologous integration decreased >10-fold. This suggests that random DNA breaks attract illegitimate recombination (IR) events that compete with homology search.     

Genetic analysis of an incomplete mutS gene from Pseudomonas putida

We genetically characterized the Pseudomonas putida mutS gene and found that it encodes a smaller MutS protein than do the genes of other bacteria. This gene is able to function in the mutS mutants of Escherichia coli and Bacillus subtilis. A P. putida mutS mutant has a mutation frequency 1,000-fold greater than that of the wild-type strain.     

Mutation of msh-2 in Neurospora crassa does not reduce the incidence of recombinants with multiple patches of donor chromosome sequence

The Neurospora homologue msh-2 of the Escherichia coli mismatch repair gene mutS was mutated by repeat-induced point mutation (RIP) of a 1.9-kb duplication covering 1661bp of the coding sequence and 302 bp 5' of the gene. msh-2(RIP-LK1) exhibited a mutator phenotype conferring a 17-fold increase in the frequency of spontaneous mitotic reversion of his-3 allele K458. In msh-2(RIP-LK1) homozygotes, recombination frequency at the his-3 locus increased up to 2.9-fold over that in msh-2(+) diploids. Progeny of crosses homozygous msh-2(RIP-LK1), like those from crosses homozygous msh-2(+) frequently had multiple patches of donor chromosome sequence, suggesting that patchiness in msh-2(+) crosses is not explained by incomplete repair of heteroduplex DNA by MSH-2. These findings are consistent with data from the analysis of events in a Neurospora translocation heterozygote that suggested multiple patches of donor chromosome sequence arising during recombination reflect multiple template switches during DNA repair synthesis.     

Mutagenesis via IS transposition in Deinococcus radiodurans

Analysis of the complete genome indicates that insertion sequences (ISs) are abundant in the radio-resistant bacterium Deinococcus radiodurans. By developing a forward mutagenesis assay to detect any inactivation events in D. radiodurans, we found that in the presence of an active mismatch repair system 75% of the mutations to trimethoprim-resistance (Tmp(R)) resulted from an IS insertion into the thyA coding region. Analysis of their distribution among the spontaneous Tmp(R) mutants indicated that five different ISs were transpositionally active. A type II Miniature Inverted-repeat Transposable Element (MITE), related to one of the deinococcal ISs, was also discovered as an insertion into thyA. Seven additional genomic copies of this MITE element were identified by BLASTN. Gamma-ray irradiation of D. radiodurans led to an increase of up to 10-fold in the frequency of Tmp(R) mutants. Analysis of the induced mutations in cells exposed to 10 kGy indicated that gamma-irradiation induced transposition of ISDra2 approximately 100-fold. A 50-fold induction of ISDra2 transposition was also observed in cells exposed to 600 J m(-2) UV-irradiation. Point mutations to rifampicin resistance (Rif(R)) were also induced by gamma-irradiation to reach a plateau at 2 kGy. The plateau value represented a 16-fold increase in the mutant frequency over the background. Although error-free repair strategies predominate in D. radiodurans, an upregulation of transposition, as well as induction of point mutations in cells recovering from DNA damage, provide a genetic variability that may have long-term evolutionary consequences on the fitness of this organism in its habitat.     

A hypermutator phenotype attenuates the virulence of Listeria monocytogenes in a mouse model

The integrity of the genetic material of bacteria is guaranteed by a set of distinct repair mechanisms. The participation of these repair systems in bacterial pathogenicity has been addressed only recently. Here, we study for the first time the participation in virulence of the MutSL mismatch repair system of Listeria monocytogenes. The mutS and mutL genes, which are contiguous in the L. monocytogenes chromosome, were identified after in silico analysis. The deduced MutS shares 62% identity with MutS of Bacillus subtilis and 50% identity with HexA, its homologue in Streptococcus pneumoniae; MutL shares 59% identity with MutL of B. subtilis and 47% identity with HexB of S. pneumoniae. Functional analysis of the mutSL locus was studied by constructing a double knock-out mutant. We showed that the deletion DeltamutSL induces: (i) a 100- to 1000-fold increase in the spontaneous mutation rate; and (ii) a 10- to 15-fold increase in the frequency of transduction, thus demonstrating the role of mutSL of L. monocytogenes in both mismatch repair and homologous recombination. We found that the deletion DeltamutSL moderately affected bacterial virulence, with a 1-log increase in the lethal dose 50% (LD50) in the mouse. Strikingly, repeated passages of the mutant strain in mice reduced virulence further. Competition assays between wild-type and mutant strains showed that the deletion DeltamutSL reduced the capacity of L. monocytogenes to survive and multiply in mice. These results thus demonstrate that, for the intracellular pathogen L. monocytogenes, a hypermutator phenotype is more deleterious than profitable to its virulence.