Accessibility of DNA polymerases to repair synthesis during nucleotide excision repair in yeast cell-free extracts.
Nucleotide excision repair (NER) removes a variety of DNA lesions. Using a yeast cell-free repair system, we have analyzed the repair synthesis step of NER. NER was proficient in yeast mutant cell-free extracts lacking DNA polymerases (Pol) beta, zeta or eta. Base excision repair was also proficient without Polbeta. Repair synthesis of NER was not affected by thermal inactivation of the temperature-sensitive mutant Polalpha (pol1-17), but was reduced after thermal inactivation of the temperature-sensitive mutant Poldelta (pol3-1) or Polvarepsilon (pol2-18). Residual repair synthesis was observed in pol3-1 and pol2-18 mutant extracts, suggesting a repair deficiency rather than a complete repair defect. Deficient NER in pol3-1 and pol2-18 mutant extracts was specifically complemented by purified yeast Poldelta and Polvarepsilon, respectively. Deleting the polymerase catalytic domain of Polvarepsilon (pol2-16) also led to a deficient repair synthesis during NER, which was complemented by purified yeast Polvarepsilon, but not by purified yeast Poleta. These results suggest that efficient repair synthesis of yeast NER requires both Poldelta and Polvarepsilon in vitro, and that the low fidelity Poleta is not accessible to repair synthesis during NER.
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306 Health Sciences Research Building, Graduate Center for Toxicology, University of Kentucky, Lexington, KY 40536, USA.
Guo
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Yuan
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Wang
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D020134:Catalytic Domain; D002474:Cell-Free System; D004249:DNA Damage; D004257:DNA Polymerase II / Q000235:genetics / Q000378:metabolism; D019951:DNA Polymerase beta / Q000235:genetics / Q000378:metabolism; D004260:DNA Repair; D004271:DNA, Fungal / Q000235:genetics / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*; D005816:Genetic Complementation Test; D009154:Mutation; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D013696:Temperature; D000099270:Y-Family DNA Polymerases
D004257:DNA Polymerase II; D019951:DNA Polymerase beta; D004271:DNA, Fungal; D004259:DNA-Directed DNA Polymerase; C425929:DNA polymerase zeta; C112338:Rad30 protein; D000099270:Y-Family DNA Polymerases