Biochemical characterization of DNA damage checkpoint complexes: clamp loader and clamp complexes with specificity for 5' recessed DNA

PLoS Biol. 2003 Nov;1(2):E33. doi: 10.1371/journal.pbio.0000033. Epub 2003 Nov 17.


The cellular pathways involved in maintaining genome stability halt cell cycle progression in the presence of DNA damage or incomplete replication. Proteins required for this pathway include Rad17, Rad9, Hus1, Rad1, and Rfc-2, Rfc-3, Rfc-4, and Rfc-5. The heteropentamer replication factor C (RFC) loads during DNA replication the homotrimer proliferating cell nuclear antigen (PCNA) polymerase clamp onto DNA. Sequence similarities suggest the biochemical functions of an RSR (Rad17-Rfc2-Rfc3-Rfc4-Rfc5) complex and an RHR heterotrimer (Rad1-Hus1-Rad9) may be similar to that of RFC and PCNA, respectively. RSR purified from human cells loads RHR onto DNA in an ATP-, replication protein A-, and DNA structure-dependent manner. Interestingly, RSR and RFC differed in their ATPase activities and displayed distinct DNA substrate specificities. RSR preferred DNA substrates possessing 5' recessed ends whereas RFC preferred 3' recessed end DNA substrates. Characterization of the biochemical loading reaction executed by the checkpoint clamp loader RSR suggests new insights into the mechanisms underlying recognition of damage-induced DNA structures and signaling to cell cycle controls. The observation that RSR loads its clamp onto a 5' recessed end supports a potential role for RHR and RSR in diverse DNA metabolism, such as stalled DNA replication forks, recombination-linked DNA repair, and telomere maintenance, among other processes.

Publication types

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

MeSH terms

  • Adenosine Triphosphate / chemistry
  • Cell Cycle Proteins / chemistry
  • Cell Line, Tumor
  • DNA / chemistry
  • DNA / ultrastructure
  • DNA Damage*
  • DNA Replication*
  • Dimerization
  • Escherichia coli / metabolism
  • Exonucleases / chemistry
  • Humans
  • Immunoprecipitation
  • Models, Biological
  • Molecular Sequence Data
  • Proliferating Cell Nuclear Antigen / metabolism*
  • Protein Binding
  • Protein Structure, Secondary
  • Recombination, Genetic*
  • Replication Protein C / chemistry*
  • Saccharomyces cerevisiae / metabolism
  • Schizosaccharomyces / metabolism
  • Schizosaccharomyces pombe Proteins / chemistry
  • Substrate Specificity
  • Telomere / ultrastructure


  • Cell Cycle Proteins
  • Proliferating Cell Nuclear Antigen
  • RFC2 protein, human
  • RFC3 protein, human
  • RFC4 protein, human
  • RFC5 protein, human
  • Rad17 protein, human
  • Schizosaccharomyces pombe Proteins
  • hus1 protein, S pombe
  • rad9 protein
  • Adenosine Triphosphate
  • DNA
  • Exonucleases
  • Rad1 protein, human
  • Replication Protein C

Associated data

  • GENBANK/AAA16121
  • GENBANK/AAA35736
  • GENBANK/AAB07268
  • GENBANK/AAB09784
  • GENBANK/AAB09786
  • GENBANK/AAB39928
  • GENBANK/AAB64700
  • GENBANK/AAB67334
  • GENBANK/AAC14138
  • GENBANK/AAC36334
  • GENBANK/AAC95526
  • GENBANK/AAH17452
  • GENBANK/CAA85036
  • GENBANK/CAA85038
  • GENBANK/CAA89596
  • GENBANK/CAA96207
  • GENBANK/CAA97907
  • GENBANK/CAA99106
  • GENBANK/CAA99434
  • GENBANK/CAA99699
  • RefSeq/NP_001265
  • RefSeq/NP_009125
  • SWISSPROT/Q13315