The Rev1 interacting region (RIR) motif in the scaffold protein XRCC1 mediates a low-affinity interaction with polynucleotide kinase/phosphatase (PNKP) during DNA single-strand break repair

J Biol Chem. 2017 Sep 29;292(39):16024-16031. doi: 10.1074/jbc.M117.806638. Epub 2017 Aug 16.


The scaffold protein X-ray repair cross-complementing 1 (XRCC1) interacts with multiple enzymes involved in DNA base excision repair and single-strand break repair (SSBR) and is important for genetic integrity and normal neurological function. One of the most important interactions of XRCC1 is that with polynucleotide kinase/phosphatase (PNKP), a dual-function DNA kinase/phosphatase that processes damaged DNA termini and that, if mutated, results in ataxia with oculomotor apraxia 4 (AOA4) and microcephaly with early-onset seizures and developmental delay (MCSZ). XRCC1 and PNKP interact via a high-affinity phosphorylation-dependent interaction site in XRCC1 and a forkhead-associated domain in PNKP. Here, we identified using biochemical and biophysical approaches a second PNKP interaction site in XRCC1 that binds PNKP with lower affinity and independently of XRCC1 phosphorylation. However, this interaction nevertheless stimulated PNKP activity and promoted SSBR and cell survival. The low-affinity interaction site required the highly conserved Rev1-interacting region (RIR) motif in XRCC1 and included three critical and evolutionarily invariant phenylalanine residues. We propose a bipartite interaction model in which the previously identified high-affinity interaction acts as a molecular tether, holding XRCC1 and PNKP together and thereby promoting the low-affinity interaction identified here, which then stimulates PNKP directly.

Keywords: DNA damage; DNA damage response; DNA repair; base excision repair (BER); oxidative stress.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Motifs
  • Amino Acid Sequence
  • Amino Acid Substitution
  • Binding Sites
  • Comet Assay
  • Conserved Sequence
  • DNA Breaks, Single-Stranded*
  • DNA Repair Enzymes / chemistry
  • DNA Repair Enzymes / genetics
  • DNA Repair Enzymes / metabolism*
  • DNA Repair*
  • DNA-Binding Proteins / chemistry
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism*
  • Humans
  • Kinetics
  • Models, Molecular*
  • Mutation
  • Oxidative Stress
  • Peptide Fragments / chemistry
  • Peptide Fragments / genetics
  • Peptide Fragments / metabolism
  • Phosphotransferases (Alcohol Group Acceptor) / chemistry
  • Phosphotransferases (Alcohol Group Acceptor) / genetics
  • Phosphotransferases (Alcohol Group Acceptor) / metabolism*
  • Protein Interaction Domains and Motifs
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / metabolism
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • Sequence Alignment
  • X-ray Repair Cross Complementing Protein 1


  • DNA-Binding Proteins
  • Peptide Fragments
  • Recombinant Fusion Proteins
  • Recombinant Proteins
  • X-ray Repair Cross Complementing Protein 1
  • XRCC1 protein, human
  • PNKP protein, human
  • Phosphotransferases (Alcohol Group Acceptor)
  • DNA Repair Enzymes