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 Pol beta. Repair synthesis of NER was not affected by thermal inactivation of the temperature-sensitive mutant Pol alpha. (pol1-17), but was reduced after thermal inactivation of the temperature-sensitive mutant Pol delta (pol3-1) or Pol epsilon (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 Pol delta and Pol epsilon, respectively. Deleting the polymerase catalytic domain of Pol epsilon (pol2-16) also led to a deficient repair synthesis during NER, which was complemented by purified yeast Pol epsilon, but not by purified yeast Pol eta. These results suggest that efficient repair synthesis of yeast NER requires both Pol delta and Pol epsilon, in vitro, and that the low fidelity Pol eta is not accessible to repair synthesis during NER.