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

Abstract

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.

Figure 1

NER in Polζ and Polη mutant extracts. (A) In vitro NER of cisplatin-damaged DNA was performed in yeast cell-free extracts of the wild-type (WT) strain CL1265-7C (lane 1) and its isogenic Polζ (rev3) deletion mutant strain AMY32 (lane 2). (B) In vitro NER of AAF-adducted DNA was performed in yeast cell-free extracts of the wild-type (WT) strain BY4741 (lane 1) and its isogenic Polη (rad30) deletion mutant strain BY4741rad30Δ (lane 2). Top, ethidium bromide-stained gel; bottom, autoradiograph of the gel. +Cisplatin and +AAF, damaged pUC18 DNA; –Cisplatin and –AAF, undamaged pGEM3Zf DNA as the internal control.

Figure 2

NER in Polβ mutant extracts. In vitro NER of AAF-adducted DNA was performed in yeast cell-free extracts of the wild-type (WT) strain TC102 (lane 1) and the Polβ (pol4) deletion mutant strain SK-2-1β (lane 2). Top, ethidium bromide-stained gel; bottom, autoradiograph of the gel. +AAF, damaged pUC18 DNA; –AAF, undamaged pGEM3Zf DNA as the internal control.

Figure 3

BER in Polβ mutant extracts. (A) In vitro BER of OsO₄-damaged DNA was performed in yeast cell-free extracts of the wild-type (WT) strain TC102 (lane 1) and the Polβ deletion mutant (pol4) strain SK-2-1β (lane 2). +OsO₄, damaged pUC18 DNA; –OsO₄, undamaged pGEM3Zf DNA as the internal control. Top, ethidium bromide-stained gel; bottom, autoradiograph of the gel. (B) In vitro BER of the uracil-containing 30mer duplex DNA was performed in yeast cell-free extracts of the wild-type (WT) strains SX46A (lane 1) and TC102 (lane 2), or in yeast cell-free extracts of the Polβ deletion mutant (pol4) strain SK-2-1β (lane 3). Repair products were separated by electrophoresis on a 20% denaturing polyacrylamide gel and visualized by autoradiography. DNA size markers in nucleotides are indicated on the right.

Figure 4

NER in temperature-sensitive mutant extracts of Polα, Polδ and Polɛ. The mutant yeast cells were grown at the permissive temperature (23°C) to late logarithmic phase of growth. The cultures were then shifted to the restrictive temperature (37°C) for 2 h. Cell-free extracts were prepared for in vitro NER. The wild-type extracts were prepared from cells grown at 37°C. In vitro NER assays were performed at 23°C for 2 h. WT, the wild-type SX46A; pol1-17, the Polα temperature-sensitive mutant 488; pol2-18, the Polɛ temperature-sensitive mutant YHA302; and pol3-1, the Polδ temperature-sensitive mutant ts370. +AAF, damaged pUC18 DNA; –AAF, undamaged pGEM3Zf DNA as the internal control. Top, ethidium bromide-stained gel; bottom, autoradiograph of the gel.

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.

Figure 6

DNA ligation during residual NER in Polδ and Polɛ mutant extracts. In vitro NER reactions were performed at 26°C for 2 h using AAF-damaged pUC18 plasmid DNA. The DNA was then purified and loaded directly onto a 1% agarose gel without prior digestion with HindIII restriction endonuclease. Electrophoresis was performed in the presence of 0.5 µg/ml ethidium bromide to separate the ligated form (CC, closed circle) of plasmid pUC18 from the unligated form (OC, opened circle). Autoradiograph of the gel is shown. WT, wild-type SX46A.

Figure 7

Deficient repair synthesis of NER in pol2-16 mutant extracts. In vitro NER was performed in yeast cell-free extracts of the wild-type strain CWY231 (lane 1) and its isogenic pol2-16 mutant strain TAY 237 (lane 2). Separately, purified yeast Polδ (0.4 U) or Polɛ (0.08 U) was added to the in vitro NER reaction for complementation, as indicated in lanes 3 and 4, respectively. After reaction, the DNA was purified and loaded directly onto a 1% agarose gel without prior digestion with HindIII restriction endonuclease. Electrophoresis was performed in the presence of 0.5 µg/ml ethidium bromide to separate the ligated forms (CC, closed circle) of the damaged pUC18 (+AAF) and the undamaged pGEM3Zf (–AAF) from the unligated forms (OC, opened circle) of the plasmids. Autoradiograph of the gel is shown.

Figure 8

Effect of purified yeast Polη on NER in pol2-16 mutant extracts. Standard in vitro NER assays were performed in the pol2-16 mutant extracts without (lane 2) or with (lane 3) 100 ng of purified yeast Polη. Lane 1, in vitro NER in yeast cell-free extract of the isogenic wild-type strain CWY231. +AAF, damaged pUC18 DNA; –AAF, undamaged pGEM3Zf DNA as the internal control. Top, ethidium bromide-stained gel; bottom, autoradiograph of the gel.