Very-Short-Patch Repair in Escherichia coli Requires the dam Adenine Methylase

Abstract

Strains of Escherichia coli which lack the dam-encoded adenine methylase are mutators due to a reduction in the efficiency of postreplication mismatch repair. In this study, we show that Dam ؊ strains are also defective in very-short-patch repair, the system which corrects T/G mismatches arising from the deamination of 5-methylcytosine. This defect is associated with decreased levels of Vsr, the endonuclease which initiates short-patch repair. We also show that production of the dcm-encoded cytosine methylase is unaffected in Dam ؊ strains. Since the dcm and vsr genes are cotranscribed, the regulation of Vsr by Dam is probably posttranscriptional.The methylation of GATC sites in the Escherichia coli K-12 genome by the dam-encoded adenine methylase is crucial for efficient methyl-directed mismatch repair (MMR) (reviewed in reference 19). MMR functions most effectively during the time period between the synthesis of a new strand of DNA and its methylation by Dam. The transient undermethylation of GATC's targets MMR to the new strand of the DNA, preserving the sequence of the template strand. The lesion itself, a mispaired or unpaired base, is recognized by MutS. In conjunction with MutL, this protein activates MutH, an endonuclease which cleaves the unmethylated strand of hemimethylated GATC sites. The repair process is completed by removal and resynthesis of the DNA between the nick and the errant base. dam strains, like mut strains, are mutators (16), characterized by an increased incidence of transition and frameshift mutations. Increased production of Dam is also mutagenic, presumably due to premature methylation of the newly synthesized DNA strand (9).In contrast to MMR, the very-short-patch (VSP) repair system of E. coli is thought to be independent of adenine methylation. VSP repair corrects T/G mismatches caused by deamination of 5-methylcytosine to thymine (reviewed in reference 12). Repair is initiated by Vsr, an endonuclease which cleaves 5Ј of the mismatched T (8). Strains lacking VSP repair have a high frequency of C-to-T mutations, primarily at CCWGG sites (W ϭ A or T). The site specificity is due to the fact that Dcm, the sole cytosine methylase of E. coli K-12, methylates the second C of this sequence. VSP repair is reduced in mutS and mutL strains but is unaffected in mutH cells (10,11,24). The independence of MutH, combined with the fact that fully methylated and unmethylated C(T/G)AGG heteroduplexes are repaired as efficiently as hemimethylated DNA, suggested that Dam methylation is not important for VSP repair. However, the extent of VSP repair in a dam background has never been tested explicitly.In this study, we used a Lac reversion assay to compare the frequency of CCAGG-to-CTAGG mutations in dam strains with that in mutS, mutL, and mutH strains. Mutation is increased far more in the dam strain than in any of the mut strains. Furthermore, the majority of mutations in the dam strain are dependent on the presence of the Dcm methylase and thus result from lack of VSP repair not from a defect in...

Epigenetic Gene Regulation in the Bacterial World

“…Synthesis of Vsr, the endonuclease that initiates VSP repair, is reduced in Dam Ϫ mutants, suggesting that Dam methylation regulates Vsr synthesis (19). The vsr gene is cotranscribed with dcm, the gene for Dcm methylase; however, synthesis of Dcm remains unaffected in a Dam Ϫ background (19). The absence of GATC sites in the dcm promoter (67) provides further evidence that Dam-mediated control of the Vsr level is not transcriptional.…”

“…Dam methylation is dispensable for VSP repair: mismatched duplexes containing GATC sites are repaired with similar efficiencies in methylated and nonmethylated DNA substrates. However, Dam Ϫ mutants of E. coli are defective in both Damdirected mismatch repair and VSP repair (19), and their VSP repair defect appears to be caused by lack of Dam methylase. Synthesis of Vsr, the endonuclease that initiates VSP repair, is reduced in Dam Ϫ mutants, suggesting that Dam methylation regulates Vsr synthesis (19).…”

“…However, Dam Ϫ mutants of E. coli are defective in both Damdirected mismatch repair and VSP repair (19), and their VSP repair defect appears to be caused by lack of Dam methylase. Synthesis of Vsr, the endonuclease that initiates VSP repair, is reduced in Dam Ϫ mutants, suggesting that Dam methylation regulates Vsr synthesis (19). The vsr gene is cotranscribed with dcm, the gene for Dcm methylase; however, synthesis of Dcm remains unaffected in a Dam Ϫ background (19).…”

Regulation ofSalmonella entericaPathogenicity Island 1 by DNA Adenine Methylation

“…The vsr gene is cotranscribed with the DNA cytosine methylase gene, dcm (Bell and Cupples 2001). In stationary cultures of E. coli Dam À mutants, Vsr synthesis is reduced while Dcm synthesis is not (Bell and Cupples 2001). Hence, differential mRNA translation and/or differential degradation of the dcm-vsr transcript may occur in Dam À hosts.…”

“…Another intriguing case involves the E. coli DNA repair endonuclease Vsr. The vsr gene is cotranscribed with the DNA cytosine methylase gene, dcm (Bell and Cupples 2001). In stationary cultures of E. coli Dam À mutants, Vsr synthesis is reduced while Dcm synthesis is not (Bell and Cupples 2001).…”