Two distinct modes of ATR activation orchestrated by Rad17 and Nbs1

Cell Rep. 2013 May 30;3(5):1651-62. doi: 10.1016/j.celrep.2013.04.018. Epub 2013 May 16.

Abstract

The ATM- and Rad3-related (ATR) kinase is a master regulator of the DNA damage response, yet how ATR is activated toward different substrates is still poorly understood. Here, we show that ATR phosphorylates Chk1 and RPA32 through distinct mechanisms at replication-associated DNA double-stranded breaks (DSBs). In contrast to the rapid phosphorylation of Chk1, RPA32 is progressively phosphorylated by ATR at Ser33 during DSB resection prior to the phosphorylation of Ser4/Ser8 by DNA-PKcs. Surprisingly, despite its reliance on ATR and TopBP1, substantial RPA32 Ser33 phosphorylation occurs in a Rad17-independent but Nbs1-dependent manner in vivo and in vitro. Importantly, the role of Nbs1 in RPA32 phosphorylation can be separated from ATM activation and DSB resection, and it is dependent upon the interaction of Nbs1 with RPA. An Nbs1 mutant that is unable to bind RPA fails to support proper recovery of collapsed replication forks, suggesting that the Nbs1-mediated mode of ATR activation is important for the repair of replication-associated DSBs.

Publication types

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

MeSH terms

  • Animals
  • Ataxia Telangiectasia Mutated Proteins / antagonists & inhibitors
  • Ataxia Telangiectasia Mutated Proteins / genetics
  • Ataxia Telangiectasia Mutated Proteins / metabolism
  • Carrier Proteins / antagonists & inhibitors
  • Carrier Proteins / genetics
  • Carrier Proteins / metabolism
  • Cell Cycle Proteins / antagonists & inhibitors
  • Cell Cycle Proteins / genetics
  • Cell Cycle Proteins / metabolism*
  • Cell Line, Tumor
  • Checkpoint Kinase 1
  • DNA Breaks, Double-Stranded
  • DNA Replication
  • DNA, Single-Stranded / genetics
  • DNA, Single-Stranded / metabolism
  • DNA-Binding Proteins / antagonists & inhibitors
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism
  • Endodeoxyribonucleases
  • HEK293 Cells
  • HeLa Cells
  • Humans
  • Nuclear Proteins / antagonists & inhibitors
  • Nuclear Proteins / genetics
  • Nuclear Proteins / metabolism*
  • Phosphorylation
  • Protein Binding
  • Protein Kinases
  • RNA Interference
  • RNA, Small Interfering / metabolism
  • Replication Protein A / metabolism

Substances

  • Carrier Proteins
  • Cell Cycle Proteins
  • DNA, Single-Stranded
  • DNA-Binding Proteins
  • NBN protein, human
  • Nuclear Proteins
  • RNA, Small Interfering
  • Rad17 protein, human
  • Replication Protein A
  • TOPBP1 protein, human
  • Protein Kinases
  • ATR protein, human
  • Ataxia Telangiectasia Mutated Proteins
  • CHEK1 protein, human
  • Checkpoint Kinase 1
  • RPA2 protein, human
  • Endodeoxyribonucleases
  • RBBP8 protein, human