TopBP1 and DNA polymerase-alpha directly recruit the 9-1-1 complex to stalled DNA replication forks

J Cell Biol. 2009 Mar 23;184(6):793-804. doi: 10.1083/jcb.200810185. Epub 2009 Mar 16.

Abstract

TopBP1 and the Rad9-Rad1-Hus1 (9-1-1) complex activate the ataxia telangiectasia mutated and Rad3-related (ATR) protein kinase at stalled replication forks. ATR is recruited to stalled forks through its binding partner, ATR-interacting protein (ATRIP); however, it is unclear how TopBP1 and 9-1-1 are recruited so that they may join ATR-ATRIP and initiate signaling. In this study, we use Xenopus laevis egg extracts to determine the requirements for 9-1-1 loading. We show that TopBP1 is required for the recruitment of both 9-1-1 and DNA polymerase (pol)-alpha to sites of replication stress. Furthermore, we show that pol-alpha is also directly required for Rad9 loading. Our study identifies an assembly pathway, which is controlled by TopBP1 and includes pol-alpha, that mediates the loading of the 9-1-1 complex onto stalled replication forks. These findings clarify early events in the assembly of checkpoint signaling complexes on DNA and identify TopBP1 as a critical sensor of replication stress.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Ataxia Telangiectasia Mutated Proteins
  • Carrier Proteins / genetics
  • Carrier Proteins / metabolism*
  • Cell Cycle Proteins / genetics
  • Cell Cycle Proteins / metabolism*
  • Chromatin / metabolism
  • Chromosomal Proteins, Non-Histone / genetics
  • Chromosomal Proteins, Non-Histone / metabolism*
  • Cloning, Molecular
  • DNA / metabolism*
  • DNA Polymerase I / genetics
  • DNA Polymerase I / metabolism*
  • DNA Replication*
  • DNA-Binding Proteins / metabolism
  • Multiprotein Complexes
  • Mutagenesis, Site-Directed
  • Protein-Serine-Threonine Kinases / metabolism
  • Signal Transduction*
  • Stress, Physiological
  • Time Factors
  • Tumor Suppressor Proteins / metabolism
  • Xenopus Proteins / genetics
  • Xenopus Proteins / metabolism*
  • Xenopus laevis

Substances

  • ATRIP protein, Xenopus
  • Carrier Proteins
  • Cell Cycle Proteins
  • Chromatin
  • Chromosomal Proteins, Non-Histone
  • DNA-Binding Proteins
  • Hus1 protein, Xenopus
  • Multiprotein Complexes
  • Rad1 protein, Xenopus
  • TopBP1 protein, Xenopus
  • Tumor Suppressor Proteins
  • Xenopus Proteins
  • rad9 protein
  • DNA
  • Atr protein, Xenopus
  • Ataxia Telangiectasia Mutated Proteins
  • Protein-Serine-Threonine Kinases
  • DNA Polymerase I