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ATP insertion opposite 8-oxo-deoxyguanosine by Pol4 mediates error-free tolerance in Schizosaccharomyces pombe.

Sastre-Moreno G, Sánchez A, Esteban V, Blanco L - Nucleic Acids Res. (2014)

Bottom Line: In cell extracts, misincorporation of ATP opposite 8oxodG was shown to be SpPol4-specific, although RNase H2 efficiently recognized the 8oxodG:AMP mispair to remove AMP and trigger error-free incorporation of dCTP.Moreover, we demonstrate that purified SpPol4 uses 8oxo-dGTP and 8oxo-GTP as substrates for DNA polymerization, although with poor efficiency compared to the incorporation of undamaged nucleotides opposite either 8oxodG or undamaged templates.This suggests that SpPol4 is specialized in tolerating 8oxodG as a DNA template, without contributing significantly to the accumulation of this lesion in the DNA.

View Article: PubMed Central - PubMed

Affiliation: Centro de Biología Molecular "Severo Ochoa" (CSIC-UAM), Universidad Autónoma, 28049 Madrid, Spain.

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Incorporation of nucleotides opposite 8oxodG by S. pombe cell extracts. (A) Gap-filling activity by WCE derived from either wild-type or Δpol4 S. pombe synchronized in G1. 20 μg of each extract were incubated with the 8oxodG-gapped substrate (2.5 nM), and the indicated amounts of either dCTP (upper panel) or dATP (lower panel). (B) Gap-filling experiments were performed as in (A) but using the indicated amounts of either CTP or ATP. After incubation at 30°C for 20 min, primer extension was analysed as described in the ‘Materials and Methods’ section.
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Figure 3: Incorporation of nucleotides opposite 8oxodG by S. pombe cell extracts. (A) Gap-filling activity by WCE derived from either wild-type or Δpol4 S. pombe synchronized in G1. 20 μg of each extract were incubated with the 8oxodG-gapped substrate (2.5 nM), and the indicated amounts of either dCTP (upper panel) or dATP (lower panel). (B) Gap-filling experiments were performed as in (A) but using the indicated amounts of either CTP or ATP. After incubation at 30°C for 20 min, primer extension was analysed as described in the ‘Materials and Methods’ section.

Mentions: To further evaluate the contribution of SpPol4 to 8oxodG tolerance in vivo, we tested the gap-filling activity of S. pombe WCE either containing or lacking SpPol4, and using the same 8oxodG-containing gap described above. It has been demonstrated that NHEJ is the predominant DSB repair mechanism during G1 phase in fission yeast (40); thus, given the possible implication of SpPol4 in this pathway (26,31), we used G1-synchronized wild-type or SpPol4-defective (Δpol4) extracts. Extracts from each strain could incorporate dCTP opposite 8oxodG (Figure 3A, top), and a second dCTP incorporation mediated by primer slippage (see also Supplementary Figure S1); however, SpPol4 contribution was not scored at any concentration (Figure 3A, top; compare wt and Δpol4 panels). The wild-type WCE also catalysed the incorporation of dATP opposite 8oxodG (Figure 3A, bottom), and a subsequent dATP incorporation mediated by strand-displacement (see also Supplementary Figure S1); however, Δpol4 WCE failed to promote misinsertion of dATP opposite 8oxodG at low concentrations (1–2.5 μM), supporting an SpPol4 contribution (Figure 3A, bottom).


ATP insertion opposite 8-oxo-deoxyguanosine by Pol4 mediates error-free tolerance in Schizosaccharomyces pombe.

Sastre-Moreno G, Sánchez A, Esteban V, Blanco L - Nucleic Acids Res. (2014)

Incorporation of nucleotides opposite 8oxodG by S. pombe cell extracts. (A) Gap-filling activity by WCE derived from either wild-type or Δpol4 S. pombe synchronized in G1. 20 μg of each extract were incubated with the 8oxodG-gapped substrate (2.5 nM), and the indicated amounts of either dCTP (upper panel) or dATP (lower panel). (B) Gap-filling experiments were performed as in (A) but using the indicated amounts of either CTP or ATP. After incubation at 30°C for 20 min, primer extension was analysed as described in the ‘Materials and Methods’ section.
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Figure 3: Incorporation of nucleotides opposite 8oxodG by S. pombe cell extracts. (A) Gap-filling activity by WCE derived from either wild-type or Δpol4 S. pombe synchronized in G1. 20 μg of each extract were incubated with the 8oxodG-gapped substrate (2.5 nM), and the indicated amounts of either dCTP (upper panel) or dATP (lower panel). (B) Gap-filling experiments were performed as in (A) but using the indicated amounts of either CTP or ATP. After incubation at 30°C for 20 min, primer extension was analysed as described in the ‘Materials and Methods’ section.
Mentions: To further evaluate the contribution of SpPol4 to 8oxodG tolerance in vivo, we tested the gap-filling activity of S. pombe WCE either containing or lacking SpPol4, and using the same 8oxodG-containing gap described above. It has been demonstrated that NHEJ is the predominant DSB repair mechanism during G1 phase in fission yeast (40); thus, given the possible implication of SpPol4 in this pathway (26,31), we used G1-synchronized wild-type or SpPol4-defective (Δpol4) extracts. Extracts from each strain could incorporate dCTP opposite 8oxodG (Figure 3A, top), and a second dCTP incorporation mediated by primer slippage (see also Supplementary Figure S1); however, SpPol4 contribution was not scored at any concentration (Figure 3A, top; compare wt and Δpol4 panels). The wild-type WCE also catalysed the incorporation of dATP opposite 8oxodG (Figure 3A, bottom), and a subsequent dATP incorporation mediated by strand-displacement (see also Supplementary Figure S1); however, Δpol4 WCE failed to promote misinsertion of dATP opposite 8oxodG at low concentrations (1–2.5 μM), supporting an SpPol4 contribution (Figure 3A, bottom).

Bottom Line: In cell extracts, misincorporation of ATP opposite 8oxodG was shown to be SpPol4-specific, although RNase H2 efficiently recognized the 8oxodG:AMP mispair to remove AMP and trigger error-free incorporation of dCTP.Moreover, we demonstrate that purified SpPol4 uses 8oxo-dGTP and 8oxo-GTP as substrates for DNA polymerization, although with poor efficiency compared to the incorporation of undamaged nucleotides opposite either 8oxodG or undamaged templates.This suggests that SpPol4 is specialized in tolerating 8oxodG as a DNA template, without contributing significantly to the accumulation of this lesion in the DNA.

View Article: PubMed Central - PubMed

Affiliation: Centro de Biología Molecular "Severo Ochoa" (CSIC-UAM), Universidad Autónoma, 28049 Madrid, Spain.

Show MeSH