Effects of photoreactivation of cyclobutane pyrimidine dimers and pyrimidine (6-4) pyrimidine 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.