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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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RNase H2-mediated 5′-incision of ribonucleotides paired to 8oxodG. (A) Ribonucleotide incision experiments performed with labelled dsDNA molecules (2.5 nM; 28mer) containing dT:dAMP, 8oxodG:dAMP,dT:AMP or 8oxodG:AMP bases pairs that were incubated separately with WCE synchronized in G1, G2 or M (30 μg). These molecules were also incubated without WCE (−), to be used as a marker of the intact substrate. A labelled oligonucleotide (Sp1C; 15mer) was used as an appropriate molecular marker. After 30 min of incubation at 30°C, incision of the DNA was analysed as described in the ‘Materials and Methods’ section. (B) Wild-type and RNH201  (Δrnh201) asynchronous WCE were incubated with labelled dsDNA molecules (2.5 nM) containing either dT:AMP, 8oxodG:AMP, dG:CMP or 8oxodG:CMP base pairs embedded, and processed as in (A). (C) Kinetics of ribonucleotide incision by RNase H2 present in wild-type G1 WCE (30 μg), performed as in (A). The percentage of 15mer product generated by RNase H2 is designated as incised DNA (%). Ribonucleotides are denoted in italics.
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Figure 5: RNase H2-mediated 5′-incision of ribonucleotides paired to 8oxodG. (A) Ribonucleotide incision experiments performed with labelled dsDNA molecules (2.5 nM; 28mer) containing dT:dAMP, 8oxodG:dAMP,dT:AMP or 8oxodG:AMP bases pairs that were incubated separately with WCE synchronized in G1, G2 or M (30 μg). These molecules were also incubated without WCE (−), to be used as a marker of the intact substrate. A labelled oligonucleotide (Sp1C; 15mer) was used as an appropriate molecular marker. After 30 min of incubation at 30°C, incision of the DNA was analysed as described in the ‘Materials and Methods’ section. (B) Wild-type and RNH201 (Δrnh201) asynchronous WCE were incubated with labelled dsDNA molecules (2.5 nM) containing either dT:AMP, 8oxodG:AMP, dG:CMP or 8oxodG:CMP base pairs embedded, and processed as in (A). (C) Kinetics of ribonucleotide incision by RNase H2 present in wild-type G1 WCE (30 μg), performed as in (A). The percentage of 15mer product generated by RNase H2 is designated as incised DNA (%). Ribonucleotides are denoted in italics.

Mentions: The high cellular concentrations of NTPs make these nucleotides valuable substrates for DNA repair and tolerance reactions; however, their persistence in the genome is undesirable as it renders DNA more susceptible to hydrolysis. In eukaryotic organisms, RNase H2 initiates the removal of single NMPs embedded within a DNA sequence (43); however, whether RNase H2 or other enzymes can initiate ribonucleotide repair of 8oxodG:AMP base pairs has not been reported yet. To evaluate this hypothesis, labelled dsDNA molecules containing a single dT:dAMP, dT:AMP, 8oxodG:dAMP or 8oxodG:AMP base pair in a central position were incubated with S. pombe cell extracts, corresponding to synchronized G1, G2 or M cell-cycle phases. None of the extracts incised the dsDNA at the central position corresponding to either dT:dAMP or 8oxodG:dAMP (Figure 5A; first and second panels), despite a reported SpMYH-like activity that could eliminate dA paired to 8oxodG (20). Conversely, the extracts efficiently incised the labelled strand, at the 5′ side of the ribo-adenosine (A in the figure), similarly in both DNA substrates containing either dT:AMP or 8oxodG:AMP base pairs (Figure 5A, third and fourth panels). Remarkably, the 15-mer incised product was better detectable in the G1 extract than in both G2 and M extracts, possibly due to higher unspecific nuclease activity in the latter.


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)

RNase H2-mediated 5′-incision of ribonucleotides paired to 8oxodG. (A) Ribonucleotide incision experiments performed with labelled dsDNA molecules (2.5 nM; 28mer) containing dT:dAMP, 8oxodG:dAMP,dT:AMP or 8oxodG:AMP bases pairs that were incubated separately with WCE synchronized in G1, G2 or M (30 μg). These molecules were also incubated without WCE (−), to be used as a marker of the intact substrate. A labelled oligonucleotide (Sp1C; 15mer) was used as an appropriate molecular marker. After 30 min of incubation at 30°C, incision of the DNA was analysed as described in the ‘Materials and Methods’ section. (B) Wild-type and RNH201  (Δrnh201) asynchronous WCE were incubated with labelled dsDNA molecules (2.5 nM) containing either dT:AMP, 8oxodG:AMP, dG:CMP or 8oxodG:CMP base pairs embedded, and processed as in (A). (C) Kinetics of ribonucleotide incision by RNase H2 present in wild-type G1 WCE (30 μg), performed as in (A). The percentage of 15mer product generated by RNase H2 is designated as incised DNA (%). Ribonucleotides are denoted in italics.
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Related In: Results  -  Collection

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Figure 5: RNase H2-mediated 5′-incision of ribonucleotides paired to 8oxodG. (A) Ribonucleotide incision experiments performed with labelled dsDNA molecules (2.5 nM; 28mer) containing dT:dAMP, 8oxodG:dAMP,dT:AMP or 8oxodG:AMP bases pairs that were incubated separately with WCE synchronized in G1, G2 or M (30 μg). These molecules were also incubated without WCE (−), to be used as a marker of the intact substrate. A labelled oligonucleotide (Sp1C; 15mer) was used as an appropriate molecular marker. After 30 min of incubation at 30°C, incision of the DNA was analysed as described in the ‘Materials and Methods’ section. (B) Wild-type and RNH201 (Δrnh201) asynchronous WCE were incubated with labelled dsDNA molecules (2.5 nM) containing either dT:AMP, 8oxodG:AMP, dG:CMP or 8oxodG:CMP base pairs embedded, and processed as in (A). (C) Kinetics of ribonucleotide incision by RNase H2 present in wild-type G1 WCE (30 μg), performed as in (A). The percentage of 15mer product generated by RNase H2 is designated as incised DNA (%). Ribonucleotides are denoted in italics.
Mentions: The high cellular concentrations of NTPs make these nucleotides valuable substrates for DNA repair and tolerance reactions; however, their persistence in the genome is undesirable as it renders DNA more susceptible to hydrolysis. In eukaryotic organisms, RNase H2 initiates the removal of single NMPs embedded within a DNA sequence (43); however, whether RNase H2 or other enzymes can initiate ribonucleotide repair of 8oxodG:AMP base pairs has not been reported yet. To evaluate this hypothesis, labelled dsDNA molecules containing a single dT:dAMP, dT:AMP, 8oxodG:dAMP or 8oxodG:AMP base pair in a central position were incubated with S. pombe cell extracts, corresponding to synchronized G1, G2 or M cell-cycle phases. None of the extracts incised the dsDNA at the central position corresponding to either dT:dAMP or 8oxodG:dAMP (Figure 5A; first and second panels), despite a reported SpMYH-like activity that could eliminate dA paired to 8oxodG (20). Conversely, the extracts efficiently incised the labelled strand, at the 5′ side of the ribo-adenosine (A in the figure), similarly in both DNA substrates containing either dT:AMP or 8oxodG:AMP base pairs (Figure 5A, third and fourth panels). Remarkably, the 15-mer incised product was better detectable in the G1 extract than in both G2 and M extracts, possibly due to higher unspecific nuclease activity in the latter.

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