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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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8oxo-dGTP and 8oxo-GTP incorporation by SpPol4 in vitro. (A) Primer extension (gap-filling) by purified GST-SpPol4 (35 nM) opposite templates dC or dA using the indicated concentrations of 8oxo-dGTP (n = 3). After incubation at 30°C for 15 min, primer extension was analysed as described in the ‘Materials and Methods’ section. (B) The same gap-filling experiment described in (A) was carried out using 8oxo-GTP (n = 3). (C) Comparative incorporation of 8oxo-dGTP/8oxo-GTP versus undamaged dGTP/GTP by purified GST-SpPol4 (35 nM) opposite the dC-gapped molecule, performed as in (A). (D) As in (C) but using dTTP and UTP as undamaged nucleotides and template dA.
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Figure 7: 8oxo-dGTP and 8oxo-GTP incorporation by SpPol4 in vitro. (A) Primer extension (gap-filling) by purified GST-SpPol4 (35 nM) opposite templates dC or dA using the indicated concentrations of 8oxo-dGTP (n = 3). After incubation at 30°C for 15 min, primer extension was analysed as described in the ‘Materials and Methods’ section. (B) The same gap-filling experiment described in (A) was carried out using 8oxo-GTP (n = 3). (C) Comparative incorporation of 8oxo-dGTP/8oxo-GTP versus undamaged dGTP/GTP by purified GST-SpPol4 (35 nM) opposite the dC-gapped molecule, performed as in (A). (D) As in (C) but using dTTP and UTP as undamaged nucleotides and template dA.

Mentions: ROS can oxidize the cellular pools of nucleotides generating 8oxo-dGTP and 8oxo-GTP, which may abound in S. pombe due to the absence of a MutT homolog and could be wrongly incorporated opposite dA. In fact, as 8oxo-dGTP is in syn configuration in solution (45), it is ready to be misrecognized (as dTTP) for most DNA polymerases. Given the flexibility of SpPol4 active site, which allows both ribo and deoxynucleotide substrates and efficiently tolerates 8oxodG template lesions, we evaluated whether SpPol4 could use 8oxo-dGTP/8oxo-GTP as nucleotides for DNA polymerization. For this, purified SpPol4 was incubated with a 5′ labelled 1nt-gapped DNA molecule with either dC or dA in the gap position and variable concentrations of either 8oxo-dGTP or 8oxo-GTP. Interestingly, SpPol4 incorporated 8oxo-dGTP with similar efficiency opposite templates dC or dA (Figure 7A), and 8oxo-GTP opposite template dC better than opposite dA, but similarly to the incorporation of 8oxo-dGTP (Figure 7B). Thus, it is tempting to speculate that the 2′OH group of the ribose is limiting polymerization specifically in the syn conformation. To better understand the incorporation of 8oxo-dGTP and 8oxo-GTP by SpPol4 during gap-filling reactions, and obtain quantitative data, we evaluated the kinetics of their insertion under steady-state conditions. This analysis demonstrated that the catalytic efficiency of 8oxo-dGTP insertion was very similar opposite either template dA or dC (Table 2). Moreover, the catalytic efficiency of 8oxo-GTP incorporation opposite dC was similar to 8oxo-dGTP insertion and 8-fold more efficient than opposite dA (Table 2).


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)

8oxo-dGTP and 8oxo-GTP incorporation by SpPol4 in vitro. (A) Primer extension (gap-filling) by purified GST-SpPol4 (35 nM) opposite templates dC or dA using the indicated concentrations of 8oxo-dGTP (n = 3). After incubation at 30°C for 15 min, primer extension was analysed as described in the ‘Materials and Methods’ section. (B) The same gap-filling experiment described in (A) was carried out using 8oxo-GTP (n = 3). (C) Comparative incorporation of 8oxo-dGTP/8oxo-GTP versus undamaged dGTP/GTP by purified GST-SpPol4 (35 nM) opposite the dC-gapped molecule, performed as in (A). (D) As in (C) but using dTTP and UTP as undamaged nucleotides and template dA.
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Figure 7: 8oxo-dGTP and 8oxo-GTP incorporation by SpPol4 in vitro. (A) Primer extension (gap-filling) by purified GST-SpPol4 (35 nM) opposite templates dC or dA using the indicated concentrations of 8oxo-dGTP (n = 3). After incubation at 30°C for 15 min, primer extension was analysed as described in the ‘Materials and Methods’ section. (B) The same gap-filling experiment described in (A) was carried out using 8oxo-GTP (n = 3). (C) Comparative incorporation of 8oxo-dGTP/8oxo-GTP versus undamaged dGTP/GTP by purified GST-SpPol4 (35 nM) opposite the dC-gapped molecule, performed as in (A). (D) As in (C) but using dTTP and UTP as undamaged nucleotides and template dA.
Mentions: ROS can oxidize the cellular pools of nucleotides generating 8oxo-dGTP and 8oxo-GTP, which may abound in S. pombe due to the absence of a MutT homolog and could be wrongly incorporated opposite dA. In fact, as 8oxo-dGTP is in syn configuration in solution (45), it is ready to be misrecognized (as dTTP) for most DNA polymerases. Given the flexibility of SpPol4 active site, which allows both ribo and deoxynucleotide substrates and efficiently tolerates 8oxodG template lesions, we evaluated whether SpPol4 could use 8oxo-dGTP/8oxo-GTP as nucleotides for DNA polymerization. For this, purified SpPol4 was incubated with a 5′ labelled 1nt-gapped DNA molecule with either dC or dA in the gap position and variable concentrations of either 8oxo-dGTP or 8oxo-GTP. Interestingly, SpPol4 incorporated 8oxo-dGTP with similar efficiency opposite templates dC or dA (Figure 7A), and 8oxo-GTP opposite template dC better than opposite dA, but similarly to the incorporation of 8oxo-dGTP (Figure 7B). Thus, it is tempting to speculate that the 2′OH group of the ribose is limiting polymerization specifically in the syn conformation. To better understand the incorporation of 8oxo-dGTP and 8oxo-GTP by SpPol4 during gap-filling reactions, and obtain quantitative data, we evaluated the kinetics of their insertion under steady-state conditions. This analysis demonstrated that the catalytic efficiency of 8oxo-dGTP insertion was very similar opposite either template dA or dC (Table 2). Moreover, the catalytic efficiency of 8oxo-GTP incorporation opposite dC was similar to 8oxo-dGTP insertion and 8-fold more efficient than opposite dA (Table 2).

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