Poly(ADP-ribose) binding to Chk1 at stalled replication forks is required for S-phase checkpoint activation

Nat Commun. 2013;4:2993. doi: 10.1038/ncomms3993.

Abstract

Damaged replication forks activate poly(ADP-ribose) polymerase 1 (PARP1), which catalyses poly(ADP-ribose) (PAR) formation; however, how PARP1 or poly(ADP-ribosyl)ation is involved in the S-phase checkpoint is unknown. Here we show that PAR, supplied by PARP1, interacts with Chk1 via a novel PAR-binding regulatory (PbR) motif in Chk1, independent of ATR and its activity. iPOND studies reveal that Chk1 associates readily with the unperturbed replication fork and that PAR is required for efficient retention of Chk1 and phosphorylated Chk1 at the fork. A PbR mutation, which disrupts PAR binding, but not the interaction with its partners Claspin or BRCA1, impairs Chk1 and the S-phase checkpoint activation, and mirrors Chk1 knockdown-induced hypersensitivity to fork poisoning. We find that long chains, but not short chains, of PAR stimulate Chk1 kinase activity. Collectively, we disclose a previously unrecognized mechanism of the S-phase checkpoint by PAR metabolism that modulates Chk1 activity at the replication fork.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 3T3 Cells
  • Amino Acid Motifs
  • Amino Acid Sequence
  • Animals
  • COS Cells
  • Cell Cycle
  • Cell Line, Tumor
  • Cell Survival
  • Checkpoint Kinase 1
  • Chlorocebus aethiops
  • Chromatin / chemistry
  • DNA Damage
  • DNA Replication
  • Green Fluorescent Proteins / metabolism
  • HEK293 Cells
  • Humans
  • Mice
  • Molecular Sequence Data
  • Mutation
  • Phosphorylation
  • Poly (ADP-Ribose) Polymerase-1
  • Poly Adenosine Diphosphate Ribose / metabolism*
  • Poly(ADP-ribose) Polymerases / metabolism*
  • Protein Binding
  • Protein Kinases / metabolism*
  • Recombinant Proteins / metabolism
  • S Phase
  • Sequence Homology, Amino Acid

Substances

  • Chromatin
  • Recombinant Proteins
  • Green Fluorescent Proteins
  • Poly Adenosine Diphosphate Ribose
  • PARP1 protein, human
  • Parp1 protein, mouse
  • Poly (ADP-Ribose) Polymerase-1
  • Poly(ADP-ribose) Polymerases
  • Protein Kinases
  • CHEK1 protein, human
  • Checkpoint Kinase 1
  • Chek1 protein, mouse