Replication-mediated DNA damage by camptothecin induces phosphorylation of RPA by DNA-dependent protein kinase and dissociates RPA:DNA-PK complexes

EMBO J. 1999 Mar 1;18(5):1397-406. doi: 10.1093/emboj/18.5.1397.

Abstract

Replication protein A (RPA) is a DNA single-strand binding protein essential for DNA replication, recombination and repair. In human cells treated with the topoisomerase inhibitors camptothecin or etoposide (VP-16), we find that RPA2, the middle-sized subunit of RPA, becomes rapidly phosphorylated. This response appears to be due to DNA-dependent protein kinase (DNA-PK) and to be independent of p53 or the ataxia telangiectasia mutated (ATM) protein. RPA2 phosphorylation in response to camptothecin required ongoing DNA replication. Camptothecin itself partially inhibited DNA synthesis, and this inhibition followed the same kinetics as DNA-PK activation and RPA2 phosphorylation. DNA-PK activation and RPA2 phosphorylation were prevented by the cell-cycle checkpoint abrogator 7-hydroxystaurosporine (UCN-01), which markedly potentiates camptothecin cytotoxicity. The DNA-PK catalytic subunit (DNA-PKcs) was found to bind RPA which was replaced by the Ku autoantigen upon camptothecin treatment. DNA-PKcs interacted directly with RPA1 in vitro. We propose that the encounter of a replication fork with a topoisomerase-DNA cleavage complex could lead to a juxtaposition of replication fork-associated RPA and DNA double-strand end-associated DNA-PK, leading to RPA2 phosphorylation which may signal the presence of DNA damage to an S-phase checkpoint mechanism.

Keywords: camptothecin/DNA damage/DNA-dependent protein kinase/RPA2 phosphorylation

MeSH terms

  • Alkaloids / pharmacology
  • Androstadienes / pharmacology
  • Antigens, Nuclear*
  • Ataxia Telangiectasia / genetics
  • Camptothecin / pharmacology*
  • Cell Cycle / genetics
  • DNA Damage / genetics*
  • DNA Helicases*
  • DNA Repair
  • DNA Replication / drug effects*
  • DNA Topoisomerases, Type I / metabolism
  • DNA-Activated Protein Kinase
  • DNA-Binding Proteins / genetics*
  • Etoposide / pharmacology
  • Humans
  • Ku Autoantigen
  • Nuclear Proteins / genetics
  • Phosphorylation
  • Protein-Serine-Threonine Kinases / genetics*
  • Replication Protein A
  • Staurosporine / analogs & derivatives
  • Tumor Cells, Cultured
  • Wortmannin

Substances

  • Alkaloids
  • Androstadienes
  • Antigens, Nuclear
  • DNA-Binding Proteins
  • Nuclear Proteins
  • RPA1 protein, human
  • Replication Protein A
  • Etoposide
  • 7-hydroxystaurosporine
  • DNA-Activated Protein Kinase
  • PRKDC protein, human
  • Protein-Serine-Threonine Kinases
  • DNA Helicases
  • XRCC5 protein, human
  • Xrcc6 protein, human
  • Ku Autoantigen
  • DNA Topoisomerases, Type I
  • Staurosporine
  • Camptothecin
  • Wortmannin