Replication protein A-mediated recruitment and activation of Rad17 complexes

Proc Natl Acad Sci U S A. 2003 Nov 25;100(24):13827-32. doi: 10.1073/pnas.2336100100. Epub 2003 Nov 6.

Abstract

The human Rad17-Rfc2-5 and Rad9-Rad1-Hus1 complexes play crucial roles in the activation of the ATR-mediated DNA damage and DNA replication stress response pathways. In response to DNA damage, Rad9 is recruited to chromatin in a Rad17-dependent manner in human cells. However, the DNA structures recognized by the Rad17-Rfc2-5 complex during the damage response have not been defined. Here, we show that replication protein A (RPA) stimulates the binding of the Rad17-Rfc2-5 complex to single-stranded DNA (ssDNA), primed ssDNA, and a gapped DNA structure. Furthermore, RPA facilitates the recruitment of the Rad9-Rad1-Hus1 complex by the Rad17-Rfc2-5 complex to primed and gapped DNA structures in vitro. These findings suggest that RPA-coated ssDNA is an important part of the structures recognized by the Rad17-Rfc2-5 complex. Unlike replication factor C (RFC), which uses the 3' primer/template junction to recruit proliferating cell nuclear antigen (PCNA), the Rad17-Rfc2-5 complex can use both the 5' and the 3' primer/template junctions to recruit the Rad9-Rad1-Hus1 complex, and it shows a preference for gapped DNA structures. These results explain how the Rad17-Rfc2-5 complex senses DNA damage and DNA replication stress to initiate checkpoint signaling.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Cell Cycle Proteins / chemistry
  • Cell Cycle Proteins / genetics
  • Cell Cycle Proteins / metabolism*
  • DNA Damage
  • DNA Replication
  • DNA, Single-Stranded / metabolism
  • DNA-Binding Proteins / chemistry
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism*
  • Humans
  • In Vitro Techniques
  • Macromolecular Substances
  • Models, Biological
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Replication Protein A
  • Replication Protein C
  • Saccharomyces cerevisiae / genetics

Substances

  • Cell Cycle Proteins
  • DNA, Single-Stranded
  • DNA-Binding Proteins
  • Macromolecular Substances
  • RFC2 protein, human
  • RFC3 protein, human
  • RPA1 protein, human
  • Rad17 protein, human
  • Recombinant Proteins
  • Replication Protein A
  • rad9 protein
  • Replication Protein C