Accelerated deamination of cytosine residues in UV-induced cyclobutane pyrimidine dimers leads to CC->TT transitions

The rate of UV-induced deamination of cytosine to uracil at a specific site in double-stranded (ds) DNA was monitored using a genetic reversion assay, M13mp2Cl41 ds DNA was exposed to 160 J/m(2) UV (254 nm), incubated al 37 degrees C, pH 7.4, for various time intervals to allow for deamination, and treated with Escherichia coli photolyase in the presence of 365 nm light to reverse cyclobutane-type pyrimidine dimers. Upon transfect-ion into uracil-glycosylase deficient (ung(-)) E. coli cells, the mutation (i.e,, reversion) frequencies in the CCCC target sequence Increased greatly with post-UV lime of incubation al 37 degrees C, nearly doubling every day that the DNA had been held at 37 degrees C. After 8 days, the reversion frequencies had increased by two orders of magnitude upon transfection into ung(-) cells, relative to isogenic ung(+) cells, indicating that most of the mutations arising in UV/photolyase-treated ds DNA were C-->T mutations mediated by a uracil intermediate. Sequencing of the revertants revealed that all mutations were single C-->T or tandem double CC-->TT mutations, An increasing percentage of tandem double CC-TT mutations was found with longer post-UV incubation times. yet none occurred if the post-UV delay lime step was omitted before photoreversal. After a 4-day delay between UV and photoreversal at 37 degrees C, greater than 84% of the total revertants had tandem double CC-TT mutations. Thus, the generation of a tandem double mutation is a time-dependent process that arises in DNA after the initial UV exposure. The rare of appearance (with a pseudo-first-order rate constant ca. 10(-6) s(-1)) of tandem double mutations during incubation of UV-irradiated DNA is inconsistent with two random, independently occurring mutational events and suggests a concerted deamination of both residues in a tandem cytosine pyrimidine (C >$($) over bar C) dimer. Considering that deamination in a C >$($) over bar C dimer occurred here with a half-life of ca. 5 days, in contrast to the measured half-life of ca, 20 000 years for spontaneous (non-UV-treated) cytosine deamination for the same target, these studies show that the formation of pyrimidine dimers in DNA increases the rate of deamination by six orders of magnitude, leading to the accelerated formation of single C-T and tandem double CC-TT mutations.