An orphan DNA (cytosine-5-)-methyltransferase in Vibrio cholerae

5-Methyl cytosine (m5C) was detected in genomic DNA of the enteric pathogen Vibrio cholerae by HPLC analysis and immunoblotting with m5C-specific antibody. Although cleavage with the restriction endonuclease EcoRII revealed the absence of a Dcm homologue in V. cholerae, analysis of the genome sequence indicated the presence of a gene, designated in this study as vchM, which encodes a DNA (cytosine-5-)-methyltransferase (m5C-MTase) designated M.Vch. M.Vch is not associated with a restriction endonuclease or a mismatch very short patch repair (Vsr)-like endonuclease and is hence an ‘orphan’ or solitary MTase, although analysis of a phylogenetic tree indicated that related cytosine MTases are all components of restriction-modification systems. M.Vch recognizes and methylates the first 5′ C in the degenerate sequence 5′-RCCGGY-3′. RT-PCR analysis suggested that vchM gene expression is increased during the stationary phase of growth. During stationary phase, the spontaneous mutation frequency in the V. cholerae wild-type strain was significantly higher than in the corresponding vchM mutant strain, suggesting that the presence of M.Vch and the absence of a very short patch (VSP) repair-like system imposes upon V. cholerae a mutator phenotype.

Interaction of MutS and Vsr: Some Dominant-NegativemutS Mutations That Disable Methyladenine-Directed Mismatch Repair Are Active in Very-Short-Patch Repair

ABSTRACT In Escherichia coli and related bacteria, the very-short-patch (VSP) repair pathway uses an endonuclease, Vsr, to correct T · G mismatches that result from the deamination of 5-methylcytosines in DNA to C · G. The products ofmutS and mutL, which are required for adenine methylation-directed mismatch repair (MMR), enhance VSP repair. Multicopy plasmids carrying mutS alleles that are dominant negative for MMR were tested for their effects on VSP repair. SomemutS mutations (class I) did not lower VSP repair in amutS + background, and most class I mutations increased VSP repair in mutS cells more than plasmids containing mutS +. Other plasmid-bornemutS mutations (class II) and mutS +decreased VSP repair in the mutS + background. Thus, MutS protein lacking functions required for MMR can still participate in VSP repair, and our results are consistent with a model in which MutS binds transiently to the mispair and then translocates away from the mispair to create a specialized structure that enhances the binding of Vsr.

Spontaneous mutation at a 5-methylcytosine hotspot is prevented by very short patch (VSP) mismatch repair.

In many strains of Escherichia coli, the product of gene dcm methylates the internal cytosines in the sequence 5'CC(A or T)GG. Spontaneous deamination of 5-methylcytosine produces thymine which, if not corrected, can result in a transition mutation. 5-Methylcytosines in the lacI gene are hotspots for spontaneous C to T mutations. dcm is linked to vsr, a gene required for very short patch (VSP) repair. VSP repair corrects T.G mispairs in the following contexts:CTAAGGGGTCC, CTTGGGGACC, TAGGGTCC and CTAGGGTC. I have investigated the relationships between cytosine methylation, mutation, and VSP repair. Spontaneous mutations in the repressor (cI) gene of lambda prophage were isolated in wild-type and mutant lysogens. A hotspot for spontaneous mutation that corresponds with a 5-methylcytosine was observed in wild-type lysogens but was not present in bacteria lacking both methylase and VSP repair activity. Introduction of a plasmid containing dcm+ and vsr+ restored the mutation hotspot. If the added plasmid carried only dcm+, the frequency of spontaneous mutations at the 5-methylcytosine was over 10-fold higher than in Dcm+Vsr+ lysogens. The addition of vsr on a plasmid to a wild-type lysogen resulted in a 4-fold reduction in mutation at the hotspot. These findings support the previously untested hypothesis that VSP repair prevents mutations resulting from deamination of 5-methylcytosine.

VERY SHORT PATCH MISMATCH REPAIR IN PHAGE LAMBDA: REPAIR SITES AND LENGTH OF REPAIR TRACTS

ABSTRACT Five amber mutations in the repressor (cl) gene of bacteriophage λ recombine anomalously with nearby cl mutations. When any of these markers is used in four-factor crosses, cl + recombinants that are expected to require three crossovers occur at high frequencies. These recombinants are attributable to very-short-patch (VSP) repair of specific mismatches in DNA heteroduplexes formed during recombination between the markers flanking cl. The sites of the repairprone mutations and the lengths of repair tracts have now been determined. Amber mutations subject to VSP repair are C to T transitions in (see PDF), the sequence methylated by the product of gene dcm, and also in the related 5′CAGG or 5′CCAG sequences. Ambers arising in CAG sequences found in other contexts, or in codons other than CAG, were not subject to VSP repair. Repair tracts rarely, if ever, exceed ten nucleotides in length, and can be as short as two nucleotides. A repair-prone mutation does not stimulate recombination between flanking cl markers.