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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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Analysis of 8oxo-dGTP and 8oxo-GTP incorporation by cell extracts. (A) Wild-type and Δpol4 G1 cell extracts (20 μg) were incubated with 1nt-gapped molecules with either a dC or dA template (2.5 nM) and the indicated concentrations of 8oxo-dGTP. After incubation at 30°C for 20 min, primer extension was analysed as described in the ‘Materials and Methods’ section. (B) Gap-filling reactions performed as in (A) but using the indicated concentrations 8oxo-GTP.
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Figure 8: Analysis of 8oxo-dGTP and 8oxo-GTP incorporation by cell extracts. (A) Wild-type and Δpol4 G1 cell extracts (20 μg) were incubated with 1nt-gapped molecules with either a dC or dA template (2.5 nM) and the indicated concentrations of 8oxo-dGTP. After incubation at 30°C for 20 min, primer extension was analysed as described in the ‘Materials and Methods’ section. (B) Gap-filling reactions performed as in (A) but using the indicated concentrations 8oxo-GTP.

Mentions: The incorporation of 8oxo-dGTP and 8oxo-GTP by wild-type and Δpol4 G1 WCE was evaluated using the 1nt-gapped molecules with either dC or dA in the gap position. The error-free incorporation of 8oxo-dGTP (opposite template dC) was very specific for SpPol4 activity at any concentration (0.5–25 μM), as incorporation was barely detectable in the Δpol4 extracts (Figure 8A, top). Conversely, other polymerases present in the WCE incorporated 8oxo-dGTP very efficiently opposite dA, precluding the detection of SpPol4 possible contribution, if any (Figure 8A, bottom). In agreement with SpPol4 unique ability to use NTPs, the incorporation of 8oxo-GTP opposite both template dC and template dA was completely SpPol4-dependent at all the concentrations tested (Figure 8B). It is worthy to note that in contrast to our previous data (Figure 7B), SpPol4 incorporated 8oxo-GTP more efficiently opposite template dA than opposite dC.


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)

Analysis of 8oxo-dGTP and 8oxo-GTP incorporation by cell extracts. (A) Wild-type and Δpol4 G1 cell extracts (20 μg) were incubated with 1nt-gapped molecules with either a dC or dA template (2.5 nM) and the indicated concentrations of 8oxo-dGTP. After incubation at 30°C for 20 min, primer extension was analysed as described in the ‘Materials and Methods’ section. (B) Gap-filling reactions performed as in (A) but using the indicated concentrations 8oxo-GTP.
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Figure 8: Analysis of 8oxo-dGTP and 8oxo-GTP incorporation by cell extracts. (A) Wild-type and Δpol4 G1 cell extracts (20 μg) were incubated with 1nt-gapped molecules with either a dC or dA template (2.5 nM) and the indicated concentrations of 8oxo-dGTP. After incubation at 30°C for 20 min, primer extension was analysed as described in the ‘Materials and Methods’ section. (B) Gap-filling reactions performed as in (A) but using the indicated concentrations 8oxo-GTP.
Mentions: The incorporation of 8oxo-dGTP and 8oxo-GTP by wild-type and Δpol4 G1 WCE was evaluated using the 1nt-gapped molecules with either dC or dA in the gap position. The error-free incorporation of 8oxo-dGTP (opposite template dC) was very specific for SpPol4 activity at any concentration (0.5–25 μM), as incorporation was barely detectable in the Δpol4 extracts (Figure 8A, top). Conversely, other polymerases present in the WCE incorporated 8oxo-dGTP very efficiently opposite dA, precluding the detection of SpPol4 possible contribution, if any (Figure 8A, bottom). In agreement with SpPol4 unique ability to use NTPs, the incorporation of 8oxo-GTP opposite both template dC and template dA was completely SpPol4-dependent at all the concentrations tested (Figure 8B). It is worthy to note that in contrast to our previous data (Figure 7B), SpPol4 incorporated 8oxo-GTP more efficiently opposite template dA than opposite dC.

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