Study of cytosine to thymine mutations at the sites ofdcm and M. Hpa II methyltransferases

It has been proposed that sites of methylation are "hotspots" for C to T mutations in bacterial and human genome. I have developed a very sensitive genetic reversion assay that quantifies the frequency of C to T mutations at various methylation sites and the repair of resulting mismatches by DNA repair processes in Escherichia coli. I have demonstrated that presence of HpaII methyltransferase (MTase) in E. coli causes a substantial increase in C to T mutations at CG sites. This is similar to the known mutagenic effects of E. coli MTase Dcm within its own recognition sequence. Using this genetic system, I have tested a homolog of an E. coli DNA repair gene in Haemophilus parainfluenzae for antimutagenic activity. Unexpectedly, the homolog was found to have little effect on the reversion frequency. Using this system, I have also shown that in vitro HpaII and SssI MTases can convert cytosine to uracil. These studies define 5-methylcytosine as an intrinsic mutagen and further elaborate the mutagenic potential of cytosine MTases.;Multi-copy clones of E. coli DNA cytosine methyltransferases (C5 MTases) Dcm and M...