Related Experiment Video

Updated: May 5, 2026

Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins
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Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins

Published on: September 28, 2012

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Biochemical characterization of DNA damage checkpoint complexes: clamp loader and clamp complexes with specificity

Viola Ellison1, Bruce Stillman

  • 1Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA.

Plos Biology
|November 19, 2003
PubMed

Insights

The Rad17-Rfc2-Rfc3-Rfc4-Rfc5 (RSR) complex loads the Rad1-Hus1-Rad9 (RHR) clamp onto DNA, distinct from Replication Factor C (RFC) and Proliferating Cell Nuclear Antigen (PCNA). RSR shows unique DNA substrate preferences, offering insights into DNA repair mechanisms.

Area of Science:

  • Molecular Biology
  • Cell Biology
  • Biochemistry

Background:

  • Cellular pathways maintain genome stability by halting cell cycle progression during DNA damage or replication stress.
  • Key proteins involved include Rad17, Rad9, Hus1, Rad1, and the Replication Factor C (RFC) complex, which loads the Proliferating Cell Nuclear Antigen (PCNA) clamp onto DNA.

Purpose of the Study:

  • To investigate the biochemical functions and DNA loading mechanisms of the RSR (Rad17-Rfc2-Rfc3-Rfc4-Rfc5) complex and its associated clamp, RHR (Rad1-Hus1-Rad9).
  • To compare the activities and substrate specificities of RSR/RHR with the known RFC/PCNA system.

Main Methods:

  • Purification of the RSR complex from human cells.
  • Biochemical assays to characterize RSR's DNA loading activity, including ATP dependence, replication protein A (RPA) dependence, and DNA structure dependence.
  • Analysis of ATPase activities and DNA substrate specificities for both RSR and RFC.

Main Results:

  • The RSR complex was purified and shown to load the RHR clamp onto DNA in a manner dependent on ATP, RPA, and DNA structure.
  • RSR and RFC exhibited distinct ATPase activities and DNA substrate preferences, with RSR favoring 5' recessed ends and RFC favoring 3' recessed ends.
  • RSR's clamp-loading mechanism provides insights into recognizing DNA damage and signaling cell cycle controls.

Conclusions:

  • The RSR complex functions as a checkpoint clamp loader, distinct from RFC.
  • RSR's preference for 5' recessed DNA ends suggests roles in DNA replication fork stalling, DNA repair, and telomere maintenance.
  • These findings elucidate novel mechanisms in genome stability and DNA damage response pathways.

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