Figure 5.

Complementation of deficient NER in pol2-18 and pol3-1 mutant cell extracts. (A) Deficient repair synthesis of NER in the pol2-18 and pol3-1 mutant extracts was complemented by adding purified yeast DNA Polα, δ or ɛ to the repair reactions as indicated. The amounts of DNA polymerases added were: Polα, 80 U; Polδ, 0.25 U; and Polɛ, 0.05 U. (B) Deficient repair synthesis of NER in the pol2-18 and pol3-1 mutant extracts was complemented by adding the purified catalytic subunit of yeast Polδ or Polɛ as indicated. The His₆-tagged catalytic subunits of Polδ (125 kDa) and Polɛ (256 kDa) were overexpressed in yeast cells and purified to near homogeneity. Both proteins were active in the polymerase activity. The amounts of proteins added were: Polδ catalytic subunit, 12 ng (+) and 20 ng (++); Polɛ catalytic subunit, 30 ng (+) and 50 ng (++). WT, the wild-type SX46A. +AAF, damaged pUC18 DNA; –AAF, undamaged pGEM3Zf DNA as the internal control. Top, ethidium bromide-stained gel; bottom, autoradiograph of the gel.