Requirement of yeast Rad1-Rad10 nuclease for the removal of 3'-blocked termini from DNA strand breaks induced by reactive oxygen species

Genes Dev. 2004 Sep 15;18(18):2283-91. doi: 10.1101/gad.1232804. Epub 2004 Sep 1.

Abstract

The Rad1-Rad10 nuclease of yeast and its human counterpart ERCC1-XPF are indispensable for nucleotide excision repair, where they act by cleaving the damaged DNA strand on the 5'-side of the lesion. Intriguingly, the ERCC1- and XPF-deficient mice show a severe postnatal growth defect and they die at approximately 3 wk after birth. Here we present genetic and biochemical evidence for the requirement of Rad1-Rad10 nuclease in the removal of 3'-blocked termini from DNA strand breaks induced on treatment of yeast cells with the oxidative DNA damaging agent H(2)O(2). Our genetic studies indicate that 3'-blocked termini are removed in yeast by the three competing pathways that involve the Apn1, Apn2, and Rad1-Rad10 nucleases, and we show that the Rad1-Rad10 nuclease proficiently cleaves DNA modified with a 3'-phosphoglycolate terminus. From these observations, we infer that deficient removal of 3'-blocking groups formed from the action of oxygen free radicals generated during normal cellular metabolism is the primary underlying cause of the inviability of apn1Delta apn2Delta rad1Delta and apn1Deltaapn2Delta rad10Delta mutants and that such a deficiency accounts also for the severe growth defects of ERCC1- and XPF-deficient mice.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Base Sequence
  • DNA Damage / physiology*
  • DNA Repair / physiology*
  • DNA Repair Enzymes
  • DNA, Fungal / metabolism
  • DNA-(Apurinic or Apyrimidinic Site) Lyase / genetics
  • DNA-(Apurinic or Apyrimidinic Site) Lyase / metabolism
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism*
  • Endodeoxyribonucleases / genetics
  • Endodeoxyribonucleases / metabolism
  • Endonucleases / genetics
  • Endonucleases / metabolism*
  • Glycolates / metabolism
  • Hydrogen Peroxide / pharmacology
  • Molecular Sequence Data
  • Mutation
  • Nucleic Acid Heteroduplexes / metabolism
  • Reactive Oxygen Species / metabolism*
  • Saccharomyces cerevisiae / drug effects
  • Saccharomyces cerevisiae / physiology*
  • Saccharomyces cerevisiae Proteins / drug effects
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / metabolism*
  • Schizosaccharomyces pombe Proteins / genetics
  • Schizosaccharomyces pombe Proteins / metabolism
  • Single-Strand Specific DNA and RNA Endonucleases
  • Transglutaminases / genetics
  • Transglutaminases / metabolism

Substances

  • DNA, Fungal
  • DNA-Binding Proteins
  • Glycolates
  • Nucleic Acid Heteroduplexes
  • RAD14 protein, S cerevisiae
  • RAD4 protein, S pombe
  • Reactive Oxygen Species
  • Saccharomyces cerevisiae Proteins
  • Schizosaccharomyces pombe Proteins
  • RAD2 protein, S cerevisiae
  • Hydrogen Peroxide
  • Transglutaminases
  • Endodeoxyribonucleases
  • Endonucleases
  • RAD1 protein, S cerevisiae
  • rad1 protein, S pombe
  • Apn1 protein, S cerevisiae
  • RAD10 protein, S cerevisiae
  • Single-Strand Specific DNA and RNA Endonucleases
  • APN2 protein, S cerevisiae
  • DNA-(Apurinic or Apyrimidinic Site) Lyase
  • DNA Repair Enzymes
  • phosphoglycolate