THE RATE OF HYDROLYTIC DEAMINATION OF 5-METHYLCYTOSINE IN DOUBLE-STRANDED DNA

The modified base, 5-methylcytosine, constitutes approximately 1% of human DNA, but sites containing 5-methylcytosine account for at least 30% of all germline and somatic point mutations. A genetic assay with a sensitivity of 1 in 10(7), based on reversion to neomycin resistance of a mutant pSV2-neo plasmid, was utilized to determine and compare the deamination rates of 5-methylcytosine and cytosine in double-stranded DNA for the first time. The rate constants for spontaneous hydrolytic deamination of 5-methylcytosine and cytosine in double-stranded DNA at 37 degrees C were 5.8 x 10(-13) s(-1) and 2.6 x 10(-13) s(-1), respectively. These rates are more than sufficient to explain the observed frequency of mutation at sites containing 5-methylcytosine and emphasize the importance of hydrolytic deamination as a major source of human mutations.