RPA Phosphorylation Inhibits DNA Resection

Mol Cell. 2019 Jul 11;75(1):145-153.e5. doi: 10.1016/j.molcel.2019.05.005. Epub 2019 May 29.


Genetic recombination in all kingdoms of life initiates when helicases and nucleases process (resect) the free DNA ends to expose single-stranded DNA (ssDNA) overhangs. Resection regulation in bacteria is programmed by a DNA sequence, but a general mechanism limiting resection in eukaryotes has remained elusive. Using single-molecule imaging of reconstituted human DNA repair factors, we identify phosphorylated RPA (pRPA) as a negative resection regulator. Bloom's syndrome (BLM) helicase together with exonuclease 1 (EXO1) and DNA2 nucleases catalyze kilobase-length DNA resection on nucleosome-coated DNA. The resulting ssDNA is rapidly bound by RPA, which further stimulates DNA resection. RPA is phosphorylated during resection as part of the DNA damage response (DDR). Remarkably, pRPA inhibits DNA resection in cellular assays and in vitro via inhibition of BLM helicase. pRPA suppresses BLM initiation at DNA ends and promotes the intrinsic helicase strand-switching activity. These findings establish that pRPA provides a feedback loop between DNA resection and the DDR.

Keywords: BLM; DNA repair; DNA2; EXO1; RPA; double-strand break; single-molecule.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Binding Sites
  • DNA Helicases / genetics
  • DNA Helicases / metabolism
  • DNA Repair Enzymes / genetics
  • DNA Repair Enzymes / metabolism
  • DNA, Single-Stranded / genetics*
  • DNA, Single-Stranded / metabolism
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Exodeoxyribonucleases / genetics
  • Exodeoxyribonucleases / metabolism
  • Feedback, Physiological*
  • Gene Expression Regulation
  • Homologous Recombination
  • Humans
  • Microscopy, Fluorescence
  • Nucleosomes / chemistry
  • Nucleosomes / metabolism
  • Oligopeptides / genetics
  • Oligopeptides / metabolism
  • Phosphorylation
  • Protein Binding
  • RecQ Helicases / genetics*
  • RecQ Helicases / metabolism
  • Recombinant Fusion Proteins / genetics*
  • Recombinant Fusion Proteins / metabolism
  • Replication Protein A / genetics*
  • Replication Protein A / metabolism
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / metabolism
  • Single Molecule Imaging


  • DNA, Single-Stranded
  • Nucleosomes
  • Oligopeptides
  • Recombinant Fusion Proteins
  • Replication Protein A
  • FLAG peptide
  • EXO1 protein, human
  • Exodeoxyribonucleases
  • Bloom syndrome protein
  • DNA Helicases
  • DNA2 protein, human
  • RecQ Helicases
  • DNA Repair Enzymes