Molecular mechanism of base excision repair of uracil-containing DNA in yeast cell-free extracts.
Base excision repair (BER) constitutes a ubiquitous excision repair mechanism, which is responsible for the removal of multiple types of damaged and inappropriate bases in DNA. We have employed a yeast cell-free system to examine the biochemical mechanism of the BER pathway in lower eukaryotes. Using uracil-containing DNA as a model substrate, we demonstrate that yeast BER requires Apn1 protein, an Escherichia coli endonuclease IV homolog. In extracts of an apn1 deletion mutant, the 5'-incision at AP (apurinic/apyrimidinic) sites is not detectable, supporting 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 repair patches predominant.
Wang
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Graduate Center for Toxicology, University of Kentucky, Lexington, Kentucky 40536, USA. zwang@pop.uky.edu
Wu
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Friedberg
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D002474:Cell-Free System; D004260:DNA Repair; D045643:DNA Repair Enzymes; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics*; D004706:Endodeoxyribonucleases / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D014498:Uracil / Q000737:chemistry*
D004271:DNA, Fungal; D029701:Saccharomyces cerevisiae Proteins; D014498:Uracil; D004706:Endodeoxyribonucleases; C083274:Apn1 protein, S cerevisiae; D045643:DNA Repair Enzymes