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Nucleic Acids Research, 2001, Vol. 29, No. 14 3123-3130
© 2001 Oxford University Press

Accessibility of DNA polymerases to repair synthesis during nucleotide excision repair in yeast cell-free extracts

Xiaohua Wu, Dongyu Guo, Fenghua Yuan and Zhigang Wang*

306 Health Sciences Research Building, Graduate Center for Toxicology, University of Kentucky, Lexington, KY 40536, USA

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) ß, {zeta} or {eta}. Base excision repair was also proficient without Polß. 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{varepsilon} (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{varepsilon}, respectively. Deleting the polymerase catalytic domain of Pol{varepsilon} (pol2-16) also led to a deficient repair synthesis during NER, which was complemented by purified yeast Pol{varepsilon}, but not by purified yeast Pol{eta}. These results suggest that efficient repair synthesis of yeast NER requires both Pol{delta} and Pol{varepsilon} in vitro, and that the low fidelity Pol{eta} is not accessible to repair synthesis during NER.

* To whom correspondence should be addressed. Tel: +1 859 323 5784; Fax: +1 859 323 1059; Email: zwang{at}pop.uky.edu


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