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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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NHEJ coupled to 8oxodG tolerance by SpPol4. (A) Scheme of SpPol4 bridging two 3′-protruding DNA molecules used for NHEJ experiments. (B) NHEJ by GST-SpPol4 (200 nM) using a labelled 3′-protruding primer molecule (5 nM) either alone or with a set of different 3′-protruding cold template molecules (12.5 nM). ATP, CTP, GTP or UTP (500 nM) were added to the reaction when indicated. After incubation at 30°C for 60 min, NHEJ was analysed as described in the ‘Materials and Methods’ section. (C) Schematic representation of the different outcomes of the NHEJ experiments shown in (B). (D) Nucleotide insertion opposite 8oxodG during NHEJ, analysed as in (B), but using the indicated amounts of either CTP or ATP. The relative efficiency of incorporation of ATP versus CTP opposite 8oxodG (20-fold higher) was quantified as described in the ‘Materials and Methods’ section. (E) Tolerance of 8oxodG during NHEJ using asynchronous WCE overexpressing either GST or GST-SpPol4 (30 μg) and physiological concentrations of ATP (3000 μM), CTP (500 μM), dATP (16 μM) and dCTP (14 μM), added as indicated. After incubation at 30°C for 30 min, NHEJ was analysed as described in the ‘Materials and Methods’ section. Ribonucleotides are denoted in italics.
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Figure 4: NHEJ coupled to 8oxodG tolerance by SpPol4. (A) Scheme of SpPol4 bridging two 3′-protruding DNA molecules used for NHEJ experiments. (B) NHEJ by GST-SpPol4 (200 nM) using a labelled 3′-protruding primer molecule (5 nM) either alone or with a set of different 3′-protruding cold template molecules (12.5 nM). ATP, CTP, GTP or UTP (500 nM) were added to the reaction when indicated. After incubation at 30°C for 60 min, NHEJ was analysed as described in the ‘Materials and Methods’ section. (C) Schematic representation of the different outcomes of the NHEJ experiments shown in (B). (D) Nucleotide insertion opposite 8oxodG during NHEJ, analysed as in (B), but using the indicated amounts of either CTP or ATP. The relative efficiency of incorporation of ATP versus CTP opposite 8oxodG (20-fold higher) was quantified as described in the ‘Materials and Methods’ section. (E) Tolerance of 8oxodG during NHEJ using asynchronous WCE overexpressing either GST or GST-SpPol4 (30 μg) and physiological concentrations of ATP (3000 μM), CTP (500 μM), dATP (16 μM) and dCTP (14 μM), added as indicated. After incubation at 30°C for 30 min, NHEJ was analysed as described in the ‘Materials and Methods’ section. Ribonucleotides are denoted in italics.

Mentions: DSBs are frequently associated with other kinds of lesions such as abasic sites, dRP residues and also damaged bases like 8oxodG (41). Thus, damage tolerance associated to DSB repair can be essential to overcome this dangerous form of DNA damage. Firstly, we wanted to know whether bona fide NHEJ could be performed in vitro by SpPol4 with NTPs, and in the absence of core factors, as recently shown for human Polμ (30). For that we used two different dsDNA molecules with 3′-protruding, partially complementary ends. After microsynapsis of the two ends, if bridged by the NHEJ polymerase, these molecules would form two 1nt gaps, one of which contains a templating dG adjacent to a connection of 4 bp of complementarity (Figure 4A). Based on previous work with human Polμ and Polλ (42), we can anticipate that a recessive 5′-phosphate exclusively present in the unlabelled DNA molecule, flanking the dG-containing gap, would orient SpPol4 to extend the labelled primer with CTP.


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)

NHEJ coupled to 8oxodG tolerance by SpPol4. (A) Scheme of SpPol4 bridging two 3′-protruding DNA molecules used for NHEJ experiments. (B) NHEJ by GST-SpPol4 (200 nM) using a labelled 3′-protruding primer molecule (5 nM) either alone or with a set of different 3′-protruding cold template molecules (12.5 nM). ATP, CTP, GTP or UTP (500 nM) were added to the reaction when indicated. After incubation at 30°C for 60 min, NHEJ was analysed as described in the ‘Materials and Methods’ section. (C) Schematic representation of the different outcomes of the NHEJ experiments shown in (B). (D) Nucleotide insertion opposite 8oxodG during NHEJ, analysed as in (B), but using the indicated amounts of either CTP or ATP. The relative efficiency of incorporation of ATP versus CTP opposite 8oxodG (20-fold higher) was quantified as described in the ‘Materials and Methods’ section. (E) Tolerance of 8oxodG during NHEJ using asynchronous WCE overexpressing either GST or GST-SpPol4 (30 μg) and physiological concentrations of ATP (3000 μM), CTP (500 μM), dATP (16 μM) and dCTP (14 μM), added as indicated. After incubation at 30°C for 30 min, NHEJ was analysed as described in the ‘Materials and Methods’ section. Ribonucleotides are denoted in italics.
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Figure 4: NHEJ coupled to 8oxodG tolerance by SpPol4. (A) Scheme of SpPol4 bridging two 3′-protruding DNA molecules used for NHEJ experiments. (B) NHEJ by GST-SpPol4 (200 nM) using a labelled 3′-protruding primer molecule (5 nM) either alone or with a set of different 3′-protruding cold template molecules (12.5 nM). ATP, CTP, GTP or UTP (500 nM) were added to the reaction when indicated. After incubation at 30°C for 60 min, NHEJ was analysed as described in the ‘Materials and Methods’ section. (C) Schematic representation of the different outcomes of the NHEJ experiments shown in (B). (D) Nucleotide insertion opposite 8oxodG during NHEJ, analysed as in (B), but using the indicated amounts of either CTP or ATP. The relative efficiency of incorporation of ATP versus CTP opposite 8oxodG (20-fold higher) was quantified as described in the ‘Materials and Methods’ section. (E) Tolerance of 8oxodG during NHEJ using asynchronous WCE overexpressing either GST or GST-SpPol4 (30 μg) and physiological concentrations of ATP (3000 μM), CTP (500 μM), dATP (16 μM) and dCTP (14 μM), added as indicated. After incubation at 30°C for 30 min, NHEJ was analysed as described in the ‘Materials and Methods’ section. Ribonucleotides are denoted in italics.
Mentions: DSBs are frequently associated with other kinds of lesions such as abasic sites, dRP residues and also damaged bases like 8oxodG (41). Thus, damage tolerance associated to DSB repair can be essential to overcome this dangerous form of DNA damage. Firstly, we wanted to know whether bona fide NHEJ could be performed in vitro by SpPol4 with NTPs, and in the absence of core factors, as recently shown for human Polμ (30). For that we used two different dsDNA molecules with 3′-protruding, partially complementary ends. After microsynapsis of the two ends, if bridged by the NHEJ polymerase, these molecules would form two 1nt gaps, one of which contains a templating dG adjacent to a connection of 4 bp of complementarity (Figure 4A). Based on previous work with human Polμ and Polλ (42), we can anticipate that a recessive 5′-phosphate exclusively present in the unlabelled DNA molecule, flanking the dG-containing gap, would orient SpPol4 to extend the labelled primer with CTP.

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
Related in: MedlinePlus