S-methylcytosine is not a mutation hot spot in nondividing Escherichia coli

Spontaneous deamination of 5-methylcytosine (5meC) causes hot spots of C . G --> T . A mutations in Escherichia coli and in human cells, In E. coli, the resulting T . G mispairs fan be corrected to C . G by very short patch (VSP) repair, which requires the product of gene vsr. Mutation hot spots in genes of replicating vsr(+) bacteria are attributable to low Vsr activity. To determine the rate of deamination of 5meC and the efficiency of VSP repair in nondividing bacteria, we used kanamycin-sensitive (Kan(S)) lysogens containing a lambda kan(-) prophage, Deamination of a 5meC in the kan(-) gene resulted in mutation to kanamycin resistance (Kan(R)). Lysogens containing a single lambda kan(-) prophage per bacterial genome were grown in synthetic medium with limiting amino acids and stored at 15 degrees C or 37 degrees C. In the absence of VSP repair, Kan(R) mutants accumulated at the rate of approximately 1.3 x 10(-7) per bacterium per day at 37 degrees C. This is similar to the 5meC --> T mutation rate reported for DNA in solution. In vsr(+) bacteria, the Kan(R) accumulation rate was 3 x 10(-9) per bacterium per day, which is not significantly higher than the rate observed when the target cytosine was unmethylated. The increase in Kan(R) mutants was barely detectable in vsr(+) cultures stored at 15 degrees C for 4 months. It is likely that mutation hot spots at 5meC in rapidly dividing cells are attributable to insufficient time for T . G correction in the interval. between deamination of 5meC and subsequent DNA replication. DNA synthesis occurred in bacteria starved for amino acids and this synthesis was not highly mutagenic.