MOLECULAR MECHANISM OF BASE EXCISION-REPAIR OF URACIL-CONTAINING DNA IN YEAST CELL-FREE-EXTRACTS

Base excision repair (BER) constitutes a ubiquitous excision repair me chanism, which is responsible for the removal of multiple types of dam aged and inappropriate bases in DNA. We have employed a yeast cell-fre e system to examine the biochemical mechanism of the BER pathway in lo wer eukaryotes. Using uracil-containing DNA as a model substrate, we d emonstrate that yeast BER requires Apn1 protein, an Escherichia coli e ndonuclease IV homolog. In extracts of an apn1 deletion mutant, the 5' -incision at AP (apurinic/apyrimidinic) sites is not detectable, suppo rting the notion that yeast contains only one major 5'-AP endonuclease . The processing of the 5'-deoxyribose phosphate moieties was found to be a rate-limiting step. During BER of uracil-containing DNA, repair patch sizes of 1-5 nucleotides were detected, with single nucleotide r epair patches predominant.