THYMINE RING SATURATION AND FRAGMENTATION PRODUCTS - LESION BYPASS, MISINSERTION AND IMPLICATIONS FOR MUTAGENESIS

We have used thymine glycol and dihydrothymine as representative ring saturation products resulting from free-radical interaction with DNA p yrimidines, and urea glycosides and beta-ureidoisobutyric acid (UBA) a s models for pyrimidine-ring fragmentation products. We have shown tha t thymine glycol and the ring-fragmentation products urea and beta-ure idoisobutyric acid, as well as abasic sites, are strong blocks to DNA polymerases in vitro. In contrast, dihydrothymine is not a block to an y of the polymerases tested. For thymine glycol, termination sites wer e observed opposite the putative lesions, whereas for the ring-fragmen tation products, the termination sites were primarily one base prior t o the lesion. These and other data have suggested that thymine glycol codes for an A, and that a base is stably inserted opposite the damage , whereas when a base is inserted opposite the non-coding lesions, it is removed by the 3 --> 5 exonuclease activity of DNA polymerase 1. De spite their efficiency as blocking lesions, thymine glycol, urea and U BA can be bypassed at low frequency in certain specific sequence conte xts. When the model lesions were introduced individually into single-s tranded biologically active DNA, we found that thymine glycol, urea, b eta-ureidoisobutyric acid, and abasic sites were all lethal lesions ha ving an activation efficiency of 1, whereas dihydrothymine was not. Th us the in vitro studies predicted the in vivo results. When the surviv al of biologically active single-stranded DNA was examined in UV-induc ed Escherichia coli cells where the block to replication was released, no increase in survival was observed for DNA containing urea or abasi c sites, suggesting inefficient bypass of these lesions. In contrast, beta-ureidoisobutyric acid survival was slightly enhanced, and transfe cting DNA containing thymine glycols was significantly reactivated. Wh en mutation induction by unique lesions was measured using f1-K12 hybr id DNA containing an E. coli target gene, thymine glycols and dihydrot hymine were found to be inefficient as premutagenic lesions, suggestin g that in vivo, as in vitro, they primarily code for A. In contrast, u rea and beta-ureidoisobutyric acid were efficient premutagenic lesions , with beta-ureidoisobutyric acid being about 4-5-fold more effective than urea glycosides, which have approximately the same rate of mutati on induction as abasic sites from purines. Sequence analysis of the mu tations resulting from these ring-fragmentation products shows that th e mutations produced are both lesion and sequence context dependent. T he possible roles that bypass efficiency and lesion-directed misinsert ion might play in mutagenesis are discussed.