DNA Methylation.

The DNA of Escherichia coli contains 19,120 6-methyladenines and 12,045 5-methylcytosines in addition to the four regular bases, and these are formed by the postreplicative action of three DNA methyltransferases. The majority of the methylated bases are formed by the Dam and Dcm methyltransferases encoded by the dam (DNA adenine methyltransferase) and dcm (DNA cytosine methyltransferase) genes. Although not essential, Dam methylation is important for strand discrimination during the repair of replication errors, controlling the frequency of initiation of chromosome replication at oriC, and the regulation of transcription initiation at promoters containing GATC sequences. In contrast, there is no known function for Dcm methylation, although Dcm recognition sites constitute sequence motifs for Very Short Patch repair of T/G base mismatches. In certain bacteria (e.g., Vibrio cholerae, Caulobacter crescentus) adenine methylation is essential, and, in C. crescentus, it is important for temporal gene expression, which, in turn, is required for coordinating chromosome initiation, replication, and division. In practical terms, Dam and Dcm methylation can inhibit restriction enzyme cleavage, decrease transformation frequency in certain bacteria, and decrease the stability of short direct repeats and are necessary for site-directed mutagenesis and to probe eukaryotic structure and function.

DNA Mismatch Repair.

DNA mismatch repair (MMR) corrects replication errors in newly synthesized DNA. It also has an antirecombination action on heteroduplexes that contain similar but not identical sequences. This review focuses on the genetics and development of MMR and not on the latest biochemical mechanisms. The main focus is on MMR in Escherichia coli, but examples from Streptococcuspneumoniae and Bacillussubtilis have also been included. In most organisms, only MutS (detects mismatches) and MutL (an endonuclease) and a single exonucleaseare present. How this system discriminates between newlysynthesized and parental DNA strands is not clear. In E. coli and its relatives, however, Dam methylation is an integral part of MMR and is the basis for strand discrimination. A dedicated site-specific endonuclease, MutH, is present, andMutL has no endonuclease activity; four exonucleases can participate in MMR. Although it might seem that the accumulated wealth of genetic and biochemical data has given us a detailed picture of the mechanism of MMR in E. coli, the existence of three competing models to explain the initiation phase indicates the complexity of the system. The mechanism of the antirecombination action of MMR is largely unknown, but only MutS and MutL appear to be necessary. A primary site of action appears to be on RecA, although subsequent steps of the recombination process can also be inhibited. In this review, the genetics of Very Short Patch (VSP) repair of T/G mismatches arising from deamination of 5-methylcytosineresidues is also discussed.

DNA Methylation.

The DNA of Escherichia coli contains 19,120 6-methyladenines and 12,045 5-methylcytosines in addition to the four regular bases, and these are formed by the postreplicative action of three DNA methyltransferases. The majority of the methylated bases are formed by the Dam and Dcmmethyltransferases encoded by the dam (DNA adenine methyltransferase) and dcm (DNA cytosine methyltransferase) genes. Although not essential, Dam methylation is important for strand discrimination during repair of replication errors, controlling the frequency of initiation of chromosome replication at oriC, and regulation of transcription initiation at promoters containing GATC sequences. In contrast, there is no known function for Dcm methylation, although Dcm recognition sites constitute sequence motifs for Very Short Patch repair of T/G base mismatches. In certain bacteria (e.g., Vibrio cholera and Caulobactercrescentus) adenine methylation is essential, and in C.crescentus it is important for temporal gene expression which, in turn, is required for coordination of chromosome initiation, replication, and division. In practical terms, Dam and Dcm methylation can inhibit restriction enzyme cleavage,decrease transformation frequency in certain bacteria,and decrease the stability of short direct repeats andare necessary for site-directed mutagenesis and to probe eukaryotic structure and function.

Dam methyltransferase is required for stable lysogeny of the Shiga toxin (Stx2)-encoding bacteriophage 933W of enterohemorrhagic Escherichia coli O157:H7.

Shiga toxin 2 (Stx2), one of the principal virulence factors of enterohemorrhagic Escherichia coli, is encoded by 933W, a lambda-like prophage. 933W prophage induction contributes to Stx2 production, and here, we provide evidence that Dam methyltransferase is essential for maintenance of 933W lysogeny. Our findings are consistent with the idea that the 933W prophage has a relatively low threshold for induction, which may promote Stx2 production during infection.

DNA mismatch repair-induced double-strand breaks.

Escherichia coli dam mutants are sensitized to the cytotoxic action of base analogs, cisplatin and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), while their mismatch repair (MMR)-deficient derivatives are tolerant to these agents. We showed previously, using pulse field gel electrophoresis (PFGE), that MMR-mediated double-strand breaks (DSBs) are produced by cisplatin in dam recB(Ts) cells at the non-permissive temperature. We demonstrate here that the majority of these DSBs require DNA replication for their formation, consistent with a model in which replication forks collapse at nicks or gaps formed during MMR. DSBs were also detected in dam recB(Ts) ada ogt cells exposed to MNNG in a dose- and MMR-dependent manner. In contrast to cisplatin, the formation of these DSBs was not affected by DNA replication and it is proposed that two separate mechanisms result in DSB formation. Replication-independent DSBs arise from overlapping base excision and MMR repair tracts on complementary strands and constitute the majority of detectable DSBs in dam recB(Ts) ada ogt cells exposed to MNNG. Replication-dependent DSBs result from replication fork collapse at O(6)-methylguanine (O(6)-meG) base pairs undergoing MMR futile cycling and are more likely to contribute to cytotoxicity. This model is consistent with the observation that fast-growing dam recB(Ts) ada ogt cells, which have more chromosome replication origins, are more sensitive to the cytotoxic effect of MNNG than the same cells growing slowly.

Increased adherence and actin pedestal formation by dam-deficient enterohaemorrhagic Escherichia coli O157:H7.

Enterohaemorrhagic Escherichia coli (EHEC) are highly infectious pathogens capable of causing severe diarrhoeal illnesses. As a critical step during their colonization, EHEC adhere intimately to intestinal epithelial cells and generate F-actin 'pedestal' structures that elevate them above surrounding cell surfaces. Intimate adhesion and pedestal formation result from delivery of the EHEC type III secretion system (TTSS) effector proteins Tir and EspF(U) into the host cell and expression of the bacterial outer membrane adhesin, intimin. To investigate a role for DNA methylation during the regulation of adhesion and pedestal formation in EHEC, we deleted the dam (DNA adenine methyltransferase) gene from EHEC O157:H7 and demonstrate that this mutation results in increased interactions with cultured host cells. EHECDeltadam exhibits dramatically elevated levels of adherence and pedestal formation when compared with wild-type EHEC, and expresses significantly higher protein levels of intimin, Tir and EspF(U). Analyses of GFP fusions, Northern blotting, reverse transcription polymerase chain reaction, and microarray experiments indicate that the abundance of Tir in the dam mutant is not due to increased transcription levels, raising the possibility that Dam methylation can indirectly control protein expression by a post-transcriptional mechanism. In contrast to other dam-deficient pathogens, EHECDeltadam is capable of robust intestinal colonization of experimentally infected animals.

Homologous recombination prevents methylation-induced toxicity in Escherichia coli.

Methylating agents such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and methyl methane sulfonate (MMS) produce a wide variety of N- and O-methylated bases in DNA, some of which can block replication fork progression. Homologous recombination is a mechanism by which chromosome replication can proceed despite the presence of lesions. The two major recombination pathways, RecBCD and RecFOR, which repair double-strand breaks (DSBs) and single-strand gaps respectively, are needed to protect against toxicity with the RecBCD system being more important. We find that recombination-deficient cell lines, such as recBCD recF, and ruvC recG, are as sensitive to the cytotoxic effects of MMS and MNNG as the most base excision repair (BER)-deficient (alkA tag) isogenic mutant strain. Recombination and BER-deficient double mutants (alkA tag recBCD) were more sensitive to MNNG and MMS than the single mutants suggesting that homologous recombination and BER play essential independent roles. Cells deleted for the polA (DNA polymerase I) or priA (primosome) genes are as sensitive to MMS and MNNG as alkA tag bacteria. Our results suggest that the mechanism of cytotoxicity by alkylating agents includes the necessity for homologous recombination to repair DSBs and single-strand gaps produced by DNA replication at blocking lesions or single-strand nicks resulting from AP-endonuclease action.

Differential effects of cisplatin and MNNG on dna mutants of Escherichia coli.

DNA mismatch repair (MMR) in mammalian cells or Escherichia coli dam mutants increases the cytotoxic effects of cisplatin and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). We found that, unlike wildtype, the dnaE486 (alpha catalytic subunit of DNA polymerase III holoenzyme) mutant, and a DnaX (clamp loader subunits) over-producer, are sensitive to cisplatin but resistant to MNNG at the permissive temperature for growth. Survival of dam-13 dnaN159 (beta sliding clamp) bacteria to cisplatin was significantly less than dam cells, suggesting decreased MMR, which may be due to reduced MutS-beta clamp interaction. We also found an elevated spontaneous mutant frequency to rifampicin resistance in dnaE486 (10-fold), dnaN159 (35-fold) and dnaX36 (10-fold) strains. The mutation spectrum in the dnaN159 strain was consistent with increased SOS induction and not indicative of MMR deficiency.

The MutS C terminus is essential for mismatch repair activity in vivo.

An Escherichia coli K-12 strain was constructed with a chromosomal deletion (mutSdelta800) in the mutS gene that produced the removal of the C-terminal 53 amino acids which are not present in the MutS crystal structure. This strain has a MutS null phenotype for mutation avoidance, anti-recombination, and sensitivity to cytotoxic agents in a dam mutant background.

Cisplatin induces DNA double-strand break formation in Escherichia coli dam mutants.

Escherichia coli dam cells are more susceptible to the cytotoxic action of cisplatin than wildtype. Dam mutS or dam mutL bacteria, however, are resistant to this agent indicating that active mismatch repair sensitizes dam cells to cisplatin toxicity. Genetic data, obtained previously, were consistent with the generation and repair of cisplatin-induced double-strand breaks (DSBs). We measured DSB formation in temperature-sensitive dam recB mutants, after exposure to cisplatin, using pulse field gel electrophoresis and observed an increase in linear 100-300 kb DNA fragments corresponding to approximately 15-45 double strand breaks per genome. The formation of these DSBs was temperature and dose-dependent and was decreased in recBC bacteria at the permissive temperature or in dam(+) or mutS control strains. There was a three-fold increase in circa 2 mb linear chromosomal fragments in dam recBC strains at the non-permissive temperature compared to recBC alone. We show that dam priA strains are not viable suggesting that DSB formation is dependent on DNA replication restart. The sensitivity of priA mutants to cisplatin is also consistent with this conclusion.

MutS inhibits RecA-mediated strand transfer with methylated DNA substrates.

DNA mismatch repair (MMR) sensitizes human and Escherichia coli dam cells to the cytotoxic action of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) while abrogation of such repair results in drug resistance. In DNA methylated by MNNG, MMR action is the result of MutS recognition of O6-methylguanine base pairs. MutS and Ada methyltransferase compete for the MNNG-induced O6-methylguanine residues, and MMR-induced cytotoxicity is abrogated when Ada is present at higher concentrations than normal. To test the hypothesis that MMR sensitization is due to decreased recombinational repair, we used a RecA-mediated strand exchange assay between homologous phiX174 substrate molecules, one of which was methylated with MNNG. MutS inhibited strand transfer on such substrates in a concentration-dependent manner and its inhibitory effect was enhanced by MutL. There was no effect of these proteins on RecA activity with unmethylated substrates. We quantified the number of O6-methylguanine residues in methylated DNA by HPLC-MS/MS and 5-10 of these residues in phiX174 DNA (5386 bp) were sufficient to block the RecA reaction in the presence of MutS and MutL. These results are consistent with a model in which methylated DNA is perceived by the cell as homeologous and prevented from recombining with homologous DNA by the MMR system.

Separation of mutation avoidance and antirecombination functions in an Escherichia coli mutS mutant.

DNA mismatch repair in Escherichia coli has been shown to be involved in two distinct processes: mutation avoidance, which removes potential mutations arising as replication errors, and antirecombination which prevents recombination between related, but not identical (homeologous), DNA sequences. We show that cells with the mutSDelta800 mutation (which removes the C-terminal 53 amino acids of MutS) on a multicopy plasmid are proficient for mutation avoidance. In interspecies genetic crosses, however, recipients with the mutSDelta800 mutation show increased recombination by up to 280-fold relative to mutS+. The MutSDelta800 protein binds to O6-methylguanine mismatches but not to intrastrand platinated GG cross-links, explaining why dam bacteria with the mutSDelta800 mutation are resistant to cisplatin, but not MNNG, toxicity. The results indicate that the C-terminal end of MutS is necessary for antirecombination and cisplatin sensitization, but less significant for mutation avoidance. The inability of MutSDelta800 to form tetramers may indicate that these are the active form of MutS.

MutS inhibits RecA-mediated strand exchange with platinated DNA substrates.

Human cell lines and Escherichia coli dam mutants are sensitive to the cytotoxic action of the anticancer agent, cisplatin. Introduction of mutations disabling DNA mismatch repair into these cell lines renders them resistant to the action of this drug. We used RecA-mediated strand exchange between homologous phiX174 molecules, one that was platinated and the other that was unmodified, to show that strand transfer is decreased in a dose-dependent manner. Transfer was severely decreased at 10 adducts per molecule (5,386 bp) and abolished with 24 adducts. At low levels of adduction, addition of MutS to the reaction further decreases the rate and yield in a dose-dependent manner. MutL addition was without effect even in the presence of MutS. The results suggest that although mismatch repair is beneficial for mutation avoidance, its antirecombination activity on inappropriate substrates can be lethal to the cell.

Spontaneous and cisplatin-induced recombination in Escherichia coli.

To measure cisplatin (cis-diaminodichloroplatinum(II))-induced recombination, we have used a qualitative intrachromosomal assay utilizing duplicate inactive lac operons containing non-overlapping deletions and selection for Lac+ recombinants. The two operons are separated by one Mb and conversion of one of them yields the Lac+ phenotype. Lac+ formation for both spontaneous and cisplatin-induced recombination requires the products of the recA, recBC, ruvA, ruvB, ruvC, priA and polA genes. Inactivation of the recF, recO, recR and recJ genes decreased cisplatin-induced, but not spontaneous, recombination. The dependence on PriA and RecBC suggests that recombination is induced following stalling or collapse of replication forks at DNA lesions to form double strand breaks. The lack of recombination induction by trans-DDP suggests that the recombinogenic lesions for cisplatin are purine-purine intrastrand crosslinks.

Regulated expression of the Escherichia coli dam gene.

Regulated expression of the Escherichia coli dam gene has been achieved with the araBAD promoter lacking a ribosome binding site. Cultures of dam mutants containing plasmid pMQ430 show no detectable methylation in the absence of arabinose and complete methylation in its presence. Dam methyltransferase is a substrate for the Lon protease.

The function of Asp70, Glu77 and Lys79 in the Escherichia coli MutH protein.

The MutH protein, which is part of the Dam-directed mismatch repair system of Escherichia coli, introduces nicks in the unmethylated strand of a hemi-methylated DNA duplex. The latent endonuclease activity of MutH is activated by interaction with MutL, another member of the repair system. The crystal structure of MutH suggested that the active site residues include Asp70, Glu77 and Lys79, which are located at the bottom of a cleft where DNA binding probably occurs. We mutated these residues to alanines and found that the mutant proteins were unable to complement a chromosomal mutH deletion. The purified mutant proteins were able to bind to DNA with a hemi-methylated GATC sequence but had no detectable endonuclease activity with or without MutL. Although the data are consistent with the prediction of a catalytic role for Asp70, Glu77 and Lys79, it cannot be excluded that they are also involved in binding to MutL.

Mutational analysis of the MutH protein from Escherichia coli.

Site-directed mutagenesis was performed on several areas of MutH based on the similarity of MutH and PvuII structural models. The aims were to identify DNA-binding residues; to determine whether MutH has the same mechanism for DNA binding and catalysis as PvuII; and to localize the residues responsible for MutH stimulation by MutL. No DNA-binding residues were identified in the two flexible loop regions of MutH, although similar loops in PvuII are involved in DNA binding. Two histidines in MutH are in a similar position as two histidines (His-84 and His-85) in PvuII that signal for DNA binding and catalysis. These MutH histidines (His-112 and His-115) were changed to alanines, but the mutant proteins had wild-type activity both in vivo and in vitro. The results indicate that the MutH signal for DNA binding and catalysis remains unknown. Instead, a lysine residue (Lys-48) was found in the first flexible loop that functions in catalysis together with the three presumed catalytic amino acids (Asp-70, Glu-77, and Lys-79). Two deletion mutations (MutHDelta224 and MutHDelta214) in the C-terminal end of the protein, localized the MutL stimulation region to five amino acids (Ala-220, Leu-221, Leu-222, Ala-223, and Arg-224).

Recombinational repair is critical for survival of Escherichia coli exposed to nitric oxide.

Nitric oxide (NO(.)) is critical to numerous biological processes, including signal transduction and macrophage-mediated immunity. In this study, we have explored the biological effects of NO(.)-induced DNA damage on Escherichia coli. The relative importance of base excision repair, nucleotide excision repair (NER), and recombinational repair in preventing NO(.)-induced toxicity was determined. E. coli strains lacking either NER or DNA glycosylases (including those that repair alkylation damage [alkA tag strain], oxidative damage [fpg nei nth strain], and deaminated cytosine [ung strain]) showed essentially wild-type levels of NO(.) resistance. However, apyrimidinic/apurinic (AP) endonuclease-deficient cells (xth nfo strain) were very sensitive to killing by NO(.), which indicates that normal processing of abasic sites is critical for defense against NO(.). In addition, recA mutant cells were exquisitely sensitive to NO(.)-induced killing. Both SOS-deficient (lexA3) and Holliday junction resolvase-deficient (ruvC) cells were very sensitive to NO(.), indicating that both SOS and recombinational repair play important roles in defense against NO(.). Furthermore, strains specifically lacking double-strand end repair (recBCD strains) were very sensitive to NO(.), which suggests that NO(.) exposure leads to the formation of double-strand ends. One consequence of these double-strand ends is that NO(.) induces homologous recombination at a genetically engineered substrate. Taken together, it is now clear that, in addition to the known point mutagenic effects of NO(.), it is also important to consider recombination events among the spectrum of genetic changes that NO(. ) can induce. Furthermore, the importance of recombinational repair for cellular survival of NO(.) exposure reveals a potential susceptibility factor for invading microbes.

The LipB protein is a negative regulator of dam gene expression in Escherichia coli.

Transcription initiation of the major promoter (P2) of the Escherichia coli dam gene increases with growth rate. The presence of three partially palindromic motifs, (TTCAGT(N(20))TGAG), designated G (growth)-boxes, within the -52 to +31 region of the promoter, may be related to growth rate control. Deletion of two of these repeats, downstream of the transcription initiation point, result in constitutive high activity of the promoter. The unlinked cde-4::miniTn10 insertion also results in severalfold higher activity of the dam P2 promoter, suggesting that this mutation resulted in the loss of a putative dam P2 repressor. The cde-4 mutation was mapped to the lipB (lipoic acid) gene, which we show encodes a 24 kDa protein initiating at a TTG codon. LipB is a highly conserved protein in animal and plant species, other bacteria, Archaea, and yeast. Plasmids expressing the native or hexahistidine-tagged LipB complement the phenotype of the cde-4 mutant strain. The level of LipB in vivo was higher in exponentially growing cells than those in the stationary phase. Three G-box motifs were also found in the lipB region. Models for the regulation of expression of the two genes are discussed.

Multiple pathways of recombination define cellular responses to cisplatin.

BACKGROUND: Cisplatin is a DNA-damaging drug used for treatment of testicular tumors. The toxicity of cisplatin probably results from its ability to form DNA adducts that inhibit polymerases. Blocked replication represents a particular challenge for tumor cells, which are committed to unremitting division. Recombination provides a mechanism by which replication can proceed despite the presence of lesions and therefore could be significant for managing cisplatin toxicity. RESULTS: Recombination-deficient Escherichia coli mutants were strikingly sensitive to cisplatin when compared with the parental strain. Our data identified both daughter-strand gap and double-strand break recombination pathways as critical for survival following treatment with cisplatin. Although it is established that nucleotide excision repair (NER) significantly protects against cisplatin toxicity, most recombination-deficient strains were as sensitive to the drug as the NER-deficient uvrA mutant. Recombination/NER deficient double mutants were more sensitive to cisplatin than the corresponding single mutants, suggesting that recombination and NER pathways play independent roles in countering cisplatin toxicity. Cisplatin was a potent recombinogen in comparison with the trans isomer and canonical alkylating agents. Mitomycin C, which like cisplatin, forms DNA cross-links, was also recombinogenic at minimally toxic doses. CONCLUSIONS: We have demonstrated that all of the major recombination pathways are critical for E. coli survival following treatment with cisplatin. Moreover, recombination pathways act independently of NER and are of equal importance to NER as genoprotective systems against cisplatin toxicity. Taken together, these results shed new light on how cells survive and succumb to this widely used anticancer drug.