ATP insertion opposite 8-oxo-deoxyguanosine by Pol4 mediates error-free tolerance in Schizosaccharomyces pombe

Nucleic Acids Res. 2014 Sep;42(15):9821-37. doi: 10.1093/nar/gku711. Epub 2014 Aug 8.

Abstract

7,8-Dihydro-8-oxo-deoxyguanosine (8oxodG) is a highly premutagenic DNA lesion due to its ability to mispair with adenine. Schizosaccharomyces pombe lacks homologs for relevant enzymes that repair 8oxodG, which suggests that this lesion could be persistent and must be tolerated. Here we show that SpPol4, the unique PolX in fission yeast, incorporates ATP opposite 8oxodG almost exclusively when all nucleotides (ribos and deoxys) are provided at physiological concentrations. Remarkably, this SpPol4-specific reaction could also occur during the NHEJ of DSBs. 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. These data are the first evidence that ribonucleotides can be used safely for 8oxodG tolerance, suggesting that insertion of the highly abundant ATP substrate could be beneficial to promote efficient and error-free repair of 8oxodG-associated DSBs. 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 8-Hydroxy-2'-Deoxyguanosine
  • Adenosine Triphosphate / metabolism*
  • Base Pair Mismatch
  • DNA End-Joining Repair
  • DNA Repair*
  • DNA-Directed DNA Polymerase / metabolism*
  • Deoxyadenine Nucleotides / metabolism
  • Deoxyguanine Nucleotides / metabolism
  • Deoxyguanosine / analogs & derivatives*
  • Deoxyguanosine / metabolism
  • Guanosine Triphosphate / analogs & derivatives
  • Guanosine Triphosphate / metabolism
  • Ribonuclease H / metabolism
  • Schizosaccharomyces / enzymology
  • Schizosaccharomyces pombe Proteins / metabolism*

Substances

  • Deoxyadenine Nucleotides
  • Deoxyguanine Nucleotides
  • Schizosaccharomyces pombe Proteins
  • 8-oxodeoxyguanosine triphosphate
  • 8-hydroxyguanosine triphosphate
  • 2'-deoxy-5'-adenosine monophosphate
  • Guanosine Triphosphate
  • 8-Hydroxy-2'-Deoxyguanosine
  • Adenosine Triphosphate
  • POL4 protein, S pombe
  • DNA-Directed DNA Polymerase
  • ribonuclease HII
  • Ribonuclease H
  • Deoxyguanosine