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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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Ribonucleotide excision repair and error-free bypass of 8oxodG using S. pombe cell extracts. The top scheme shows the DNA substrate used, and the order of events compatible with the ribonucleotide excision repair (RER) pathway. Wild-type and Δpol4 WCE synchronized in G1 (30 μg) were incubated with the labelled dsDNA molecule containing the 8oxodG:AMP base and. ATP (3000 μM), CTP (500 μM), dATP (16 μM), dCTP (14 μM), dGTP (12 μM) or dTTP (30 μM) were added to the reaction when indicated. After 30 min at 30°C, incision and further extension was analysed as described in the ‘Materials and Methods’ section. M1, labelled oligonucleotide (Sp1C; 15mer) used as a molecular marker. M2, labelled dsDNA molecule (28mer) used as substrate for RER. In the figure, ribonucleotides are denoted in italics.
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Figure 6: Ribonucleotide excision repair and error-free bypass of 8oxodG using S. pombe cell extracts. The top scheme shows the DNA substrate used, and the order of events compatible with the ribonucleotide excision repair (RER) pathway. Wild-type and Δpol4 WCE synchronized in G1 (30 μg) were incubated with the labelled dsDNA molecule containing the 8oxodG:AMP base and. ATP (3000 μM), CTP (500 μM), dATP (16 μM), dCTP (14 μM), dGTP (12 μM) or dTTP (30 μM) were added to the reaction when indicated. After 30 min at 30°C, incision and further extension was analysed as described in the ‘Materials and Methods’ section. M1, labelled oligonucleotide (Sp1C; 15mer) used as a molecular marker. M2, labelled dsDNA molecule (28mer) used as substrate for RER. In the figure, ribonucleotides are denoted in italics.

Mentions: Ribonucleotide excision repair (RER), reconstituted in vitro with enzymes purified from S. cerevisiae, requires several steps: RNase H2 incision (5′ of the ribonucleotide), strand displacement-mediated DNA polymerization by Pol∂ and PCNA, and finally flap excision and DNA ligation performed by FEN1 and DNA ligase I, respectively (44). In an attempt to reproduce the steps of RER that could be excising ribonucleotides inserted opposite 8oxodG lesions, and could require SpPol4 activity, we used the dsDNA molecule containing an 8oxodG:AMP base pair (Figure 6; see scheme), physiological concentrations of selected nucleotides, and either wild-type or Δpol4 WCE (synchronized in G1). Following the activity of RNase H2, some ATP could be incorporated by the wild-type extract but barely by the pol4-deficient (Figure 6, lanes 2 versus 10), demonstrating again that although this reaction is limited, it is SpPol4-dependent; however, if ATP is reinserted during RER, it would be unproductive, as it would restore the excised ribonucleotide. Neither CTP nor dATP (two valid SpPol4 substrates to be inserted opposite 8oxodG, as shown in vitro) were incorporated by the extracts (Figure 6, lanes 3–4 and 11–12). Remarkably, dCTP was inserted as efficiently as ATP opposite 8oxodG, but irrespective of the presence of SpPol4 (Figure 6, lanes 5 and 13), demonstrating that the excised ribonucleotide can be substituted by dC, thus triggering error-free tolerance of this lesion.


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)

Ribonucleotide excision repair and error-free bypass of 8oxodG using S. pombe cell extracts. The top scheme shows the DNA substrate used, and the order of events compatible with the ribonucleotide excision repair (RER) pathway. Wild-type and Δpol4 WCE synchronized in G1 (30 μg) were incubated with the labelled dsDNA molecule containing the 8oxodG:AMP base and. ATP (3000 μM), CTP (500 μM), dATP (16 μM), dCTP (14 μM), dGTP (12 μM) or dTTP (30 μM) were added to the reaction when indicated. After 30 min at 30°C, incision and further extension was analysed as described in the ‘Materials and Methods’ section. M1, labelled oligonucleotide (Sp1C; 15mer) used as a molecular marker. M2, labelled dsDNA molecule (28mer) used as substrate for RER. In the figure, ribonucleotides are denoted in italics.
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Related In: Results  -  Collection

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Figure 6: Ribonucleotide excision repair and error-free bypass of 8oxodG using S. pombe cell extracts. The top scheme shows the DNA substrate used, and the order of events compatible with the ribonucleotide excision repair (RER) pathway. Wild-type and Δpol4 WCE synchronized in G1 (30 μg) were incubated with the labelled dsDNA molecule containing the 8oxodG:AMP base and. ATP (3000 μM), CTP (500 μM), dATP (16 μM), dCTP (14 μM), dGTP (12 μM) or dTTP (30 μM) were added to the reaction when indicated. After 30 min at 30°C, incision and further extension was analysed as described in the ‘Materials and Methods’ section. M1, labelled oligonucleotide (Sp1C; 15mer) used as a molecular marker. M2, labelled dsDNA molecule (28mer) used as substrate for RER. In the figure, ribonucleotides are denoted in italics.
Mentions: Ribonucleotide excision repair (RER), reconstituted in vitro with enzymes purified from S. cerevisiae, requires several steps: RNase H2 incision (5′ of the ribonucleotide), strand displacement-mediated DNA polymerization by Pol∂ and PCNA, and finally flap excision and DNA ligation performed by FEN1 and DNA ligase I, respectively (44). In an attempt to reproduce the steps of RER that could be excising ribonucleotides inserted opposite 8oxodG lesions, and could require SpPol4 activity, we used the dsDNA molecule containing an 8oxodG:AMP base pair (Figure 6; see scheme), physiological concentrations of selected nucleotides, and either wild-type or Δpol4 WCE (synchronized in G1). Following the activity of RNase H2, some ATP could be incorporated by the wild-type extract but barely by the pol4-deficient (Figure 6, lanes 2 versus 10), demonstrating again that although this reaction is limited, it is SpPol4-dependent; however, if ATP is reinserted during RER, it would be unproductive, as it would restore the excised ribonucleotide. Neither CTP nor dATP (two valid SpPol4 substrates to be inserted opposite 8oxodG, as shown in vitro) were incorporated by the extracts (Figure 6, lanes 3–4 and 11–12). Remarkably, dCTP was inserted as efficiently as ATP opposite 8oxodG, but irrespective of the presence of SpPol4 (Figure 6, lanes 5 and 13), demonstrating that the excised ribonucleotide can be substituted by dC, thus triggering error-free tolerance of this lesion.

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