Martin G. Marinus

The dam and dcm strains of Escherichia coli--a review

The construction of a variety of strains deficient in the methylation of adenine and cytosine residues in DNA by the methyltransferases (MTases) Dam and Dcm has allowed the study of the role of these enzymes in the biology of Escherichia coli. Dam methylation has been shown to play a role in coordinating DNA replication initiation, DNA mismatch repair and the regulation of expression of some genes. The regulation of expression of dam has been found to be complex and influenced by five promoters. A role for Dcm methylation in the cell remains elusive and dcm- cells have no obvious phenotype. dam- and dcm- strains have a range of uses in molecular biology and bacterial genetics, including preparation of DNA for restriction by some restriction endonucleases, for transformation into other bacterial species, nucleotide sequencing and site-directed mutagenesis. A variety of assays are available for rapid detection of both the Dam and Dcm phenotypes. A number of restriction systems in E. coli have been described which recognise foreign DNA methylation, but ignore Dam and Dcm methylation. Here, we describe the most commonly used mutant alleles of dam and dcm and the characteristics of a variety of the strains that carry these genes. A description of several plasmids that carry dam gene constructs is also included.

Escherichia coli mutator genes

The isolation and characterization of Escherichia coli mutator genes have led to a better understanding of DNA replication fidelity mechanisms and to the discovery of important DNA repair pathways and their relationship to spontaneous mutagenesis. Mutator strains in a population of cells can be beneficial in that they allow rapid selection of variants during periods of stress, such as drug exposure.

Mismatch correction at O6-methylguanine residues in E. coli DNA

Escherichia coli has a correction system which removes mismatched bases from DNA1. Mutants (dam) which lack the major DNA adenine methylase2 are hypersensitive to the effects of base analogue mutagens such as 2-aminopurine3 and appear to be defective in mismatch correction. Phenotypic revertants of dam mutants to base analogue resistance include second site mutations in mutL or mutS genes4, which are also part of the correction system. We report here that E. coli dam mutants are also sensitive to the DNA methylating agent N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), which introduces O6-methylguanine (m6G) into the DNA. This sensitivity, however, was not observed using methylating agents which generate only low amounts of this alkylated base. Furthermore, the introduction of either a mutL or a mutS mutation into dam strains abolished the sensitivity to MNNG. These results suggest that mismatch correction occurs at m6G residues in DNA. These lesions miscode in DNA polymerase I-mediated DNA synthesis in vitro5 and are known to be mutagenic in vivo6. Nevertheless, it appears that mismatch correction at m6G residues in DNA does not lead to reduced induction of mutation by MNNG.

Biological function for 6-methyladenine residues in the DNA of Escherichia coli K12☆

Abstract A strain of Escherichia coli K12 mutant at the dam † site contains 0.8 mole % 6-methyl adenine as compared to 0.50 mole % in the wild type, and the residual DNA methylation is not due to the K12 modification methylase specified by the hsp genes. The dam-3 mutant is more sensitive to ultraviolet irradiation and to mitomycin C than the wild type and also shows a higher mutability. DNA isolated from the dam-3 mutant contains single-stranded breaks that are amplified in dam-3 polA12 and dam-3 lig-7 double mutants. A function of dam -specified 6-methyl adenine residues in DNA would, therefore, appear to be the protection of DNA from a nuclease(s) that causes the development of breaks. Combination of dam-3 with polA, recA, recB and recC is lethal.

Location of DNA methylation genes on the Escherichia coli K-12 genetic map

SummaryThe genes responsible for DNA adenine methylation (dam) and DNA cytosine methylation (dcm) have been mapped on the E. coli K-12 genetic map. The dam gene is situated at min 65 and the gene order cysG-(trpS, dam)-aro B inferred. The dcm gene is located at min 37.5 and the gene order is supD-dcm-flaA1. In F′ merodiploids, the dam and dcm alleles are recessive.

Pleiotropic effects of a DNA adenine methylation mutation (dam-3) in Escherichia coli K12

The dam-3 mutation results in a five-fold reduction in the number of 6-methyl-adenine (6-meA) residues in the DNA of E. coli K12 or phage lambda. The DNA of phage fd appears to be devoid of 6-meA when propagated on dam-3 bacteria. The phenotypic differences between dam-3 and dam+ bacteria include: (i) increased free phage in lysogenic dam-3 cultures, (2) increased sensitivity to methyl methanesulfonate (MMS), (3) inviability of dam-3 lex-I strains, (4) lower molecular weight of DNA in dam-3 bacteria in the absence of DNA ligase and (5) increased rate of DNA degradation in dam-3 recA strains.

Role of SeqA and Dam in Escherichia coli gene expression: A global/microarray analysis

High-density oligonucleotide arrays were used to monitor global transcription patterns in Escherichia coli with various levels of Dam and SeqA proteins. Cells lacking Dam methyltransferase showed a modest increase in transcription of the genes belonging to the SOS regulon. Bacteria devoid of the SeqA protein, which preferentially binds hemimethylated DNA, were found to have a transcriptional profile almost identical to WT bacteria overexpressing Dam methyltransferase. The latter two strains differed from WT in two ways. First, the origin proximal genes were transcribed with increased frequency due to increased gene dosage. Second, chromosomal domains of high transcriptional activity alternate with regions of low activity, and our results indicate that the activity in each domain is modulated in the same way by SeqA deficiency or Dam overproduction. We suggest that the methylation status of the cell is an important factor in forming and/or maintaining chromosome structure.

Correlation of DNA adenine methylase activity with spontaneous mutability in Escherichia coli K-12

Using a multicopy plasmid in which the tac promoter has been placed in front of the dam gene of Escherichia coli K-12, we show that levels of DNA adenine methylase activity are correlated with the spontaneous mutation frequency.

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.

Insertion Mutations in the dam Gene of Escherichia coli K-12

SummaryThe dam gene of E. coli can be inactivated by insertion of Tn9 or Mud phage. Strains bearing these mutations are viable indicating that the dam gene product is dispensable.