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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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Error-free versus error-prone incorporation opposite 8oxodG by SpPol4 in vitro. (A) Scheme of the labelled substrates used to determine the gap-filling activity of SpPol4 in vitro. The molecules contained a 1nt-gap (X) with 8oxodG, dG or dT in the gap position and a recessive 5′-phosphate flanking the gap. (B) Primer extension (gap-filling) by purified GST-SpPol4 (35 nM), templated either by 8oxodG, dT or dG, with the indicated amounts of dCTP or dATP (n = 3). After incubation at 30°C for 15 min, samples were processed as described in the ‘Materials and Methods’ section. (C) The same gap-filling analysis performed in (B) but using the indicated amounts of either ATP or CTP (n = 3).
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Figure 1: Error-free versus error-prone incorporation opposite 8oxodG by SpPol4 in vitro. (A) Scheme of the labelled substrates used to determine the gap-filling activity of SpPol4 in vitro. The molecules contained a 1nt-gap (X) with 8oxodG, dG or dT in the gap position and a recessive 5′-phosphate flanking the gap. (B) Primer extension (gap-filling) by purified GST-SpPol4 (35 nM), templated either by 8oxodG, dT or dG, with the indicated amounts of dCTP or dATP (n = 3). After incubation at 30°C for 15 min, samples were processed as described in the ‘Materials and Methods’ section. (C) The same gap-filling analysis performed in (B) but using the indicated amounts of either ATP or CTP (n = 3).

Mentions: To elucidate a possible role of SpPol4 in the tolerance of 8oxodG, we first evaluated its efficiency and preference of incorporation of dNTPs opposite the lesion. For this, purified SpPol4 was assayed on a 1nt­-gapped DNA molecule either with dG, 8oxodG, or dT in the gap position (X in Figure 1A). This DNA molecule, that mimics BER and NHEJ intermediates, has a recessive 5′-phosphate flanking the gap, which increases the efficiency of DNA polymerization by purified SpPol4 (26). As shown in Figure 1B, SpPol4 could incorporate dCTP and dATP opposite 8oxodG but, strikingly, it performed the error-prone reaction (8oxodG:dAMP) with much higher efficiency. Moreover, incorporation of dATP opposite 8oxodG was only around 3-fold less efficient than opposite dT, and similar to the insertion of dCTP opposite dG. Likewise other PolXs, such as Polμ and TdT, SpPol4 has the extraordinary ability to incorporate NTPs to a DNA primer (26). Remarkably, SpPol4 could tolerate 8oxodG using NTPs (Figure 1C), and as efficiently as using dNTPs. Again, SpPol4 displayed a similar preference for the error-prone reaction (8oxodG:AMP), that also reached the efficiency of CTP incorporation opposite an undamaged dG (dG:CMP; Figure 1C).


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)

Error-free versus error-prone incorporation opposite 8oxodG by SpPol4 in vitro. (A) Scheme of the labelled substrates used to determine the gap-filling activity of SpPol4 in vitro. The molecules contained a 1nt-gap (X) with 8oxodG, dG or dT in the gap position and a recessive 5′-phosphate flanking the gap. (B) Primer extension (gap-filling) by purified GST-SpPol4 (35 nM), templated either by 8oxodG, dT or dG, with the indicated amounts of dCTP or dATP (n = 3). After incubation at 30°C for 15 min, samples were processed as described in the ‘Materials and Methods’ section. (C) The same gap-filling analysis performed in (B) but using the indicated amounts of either ATP or CTP (n = 3).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
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Figure 1: Error-free versus error-prone incorporation opposite 8oxodG by SpPol4 in vitro. (A) Scheme of the labelled substrates used to determine the gap-filling activity of SpPol4 in vitro. The molecules contained a 1nt-gap (X) with 8oxodG, dG or dT in the gap position and a recessive 5′-phosphate flanking the gap. (B) Primer extension (gap-filling) by purified GST-SpPol4 (35 nM), templated either by 8oxodG, dT or dG, with the indicated amounts of dCTP or dATP (n = 3). After incubation at 30°C for 15 min, samples were processed as described in the ‘Materials and Methods’ section. (C) The same gap-filling analysis performed in (B) but using the indicated amounts of either ATP or CTP (n = 3).
Mentions: To elucidate a possible role of SpPol4 in the tolerance of 8oxodG, we first evaluated its efficiency and preference of incorporation of dNTPs opposite the lesion. For this, purified SpPol4 was assayed on a 1nt­-gapped DNA molecule either with dG, 8oxodG, or dT in the gap position (X in Figure 1A). This DNA molecule, that mimics BER and NHEJ intermediates, has a recessive 5′-phosphate flanking the gap, which increases the efficiency of DNA polymerization by purified SpPol4 (26). As shown in Figure 1B, SpPol4 could incorporate dCTP and dATP opposite 8oxodG but, strikingly, it performed the error-prone reaction (8oxodG:dAMP) with much higher efficiency. Moreover, incorporation of dATP opposite 8oxodG was only around 3-fold less efficient than opposite dT, and similar to the insertion of dCTP opposite dG. Likewise other PolXs, such as Polμ and TdT, SpPol4 has the extraordinary ability to incorporate NTPs to a DNA primer (26). Remarkably, SpPol4 could tolerate 8oxodG using NTPs (Figure 1C), and as efficiently as using dNTPs. Again, SpPol4 displayed a similar preference for the error-prone reaction (8oxodG:AMP), that also reached the efficiency of CTP incorporation opposite an undamaged dG (dG:CMP; Figure 1C).

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