Effects of photoreactivation of cyclobutane pyrimidine dimers and pyrimidine (6-4) pyrimidone photoproducts on ultraviolet mutagenesis in SOS-induced repair-deficient Escherichia coli.

Abstract

Using purified photolyases for pyrimidine (6-4) pyrimidone photoproducts [(6-4)PP] and cyclobutane pyrimidine dimers (CPD), the effects of photoreactivation on mutagenesis were examined in the supF gene on a plasmid transfected into repair-deficient SOS-induced Escherichia coli host cells. More than 95% of CPD and (6-4)PP were removed from plasmid DNA by treatment with CPD photolyase and (6-4)photolyase, respectively. In each photolyase treatment, base substitutions at dipyrimidine sequences were predominantly observed. Of the single base substitutions observed after CPD photoreactivation, 83% were A:T-->G:C transitions at 5'-TT-3' sites. After (6-4)photolyase treatment, 81% were G:C-->A:T transitions at 5'-CC-3' and 5'-TC-3' sequences. Thus, the major mutagenic photoproducts of single-base substitutions were CPD at 5'-CC-3' or 5'-TC-3' sites and (6-4)PP at 5'-TT-3' sites. Tandem double mutations occurred mainly at 5'-CC-3' sites and were CPD-photoreactivated, suggesting that CPD at 5'-CC-3' was responsible for tandem double mutations. After photoreactivation of both CPD and (6-4)PP, single-base substitutions were primarily G:C-->A:T transitions at 5'-CC-3' or 5'-TC-3' sites and A:T-->G:C transitions at 5'-TT-3' sites, and secondarily G:C-->T:A transversions at 5'-CC-3' sites, G:C-->C:G transversions at 5'-CC-3' sites and A:T-->T:A transversions at 5'-TT-3' sites, which were essentially the same as those observed after photoreactivation of CPD alone, (6-4)PP alone and without photoreactivation. Thus, these transversions were not derived from unknown UV adducts but from incompletely repaired CPD and (6-4)PP.