Purification and characterization of human DNA damage checkpoint Rad complexes.
Checkpoint Rad proteins function early in the DNA damage checkpoint signaling cascade to arrest cell cycle progression in response to DNA damage. This checkpoint ensures the transmission of an intact genetic complement to daughter cells. To learn about the damage sensor function of the human checkpoint Rad proteins, we purified a heteropentameric complex composed of hRad17-RFCp36-RFCp37-RFCp38-RFCp40 (hRad17-RFC) and a heterotrimeric complex composed of hRad9-hHus1-hRad1 (checkpoint 9-1-1 complex). hRad17-RFC binds to DNA, with a preference for primed DNA and possesses weak ATPase activity that is stimulated by primed DNA and single-stranded DNA. hRad17-RFC forms a complex with the 9-1-1 heterotrimer reminiscent of the replication factor C/proliferating cell nuclear antigen clamp loader/sliding clamp complex of the replication machinery. These findings constitute biochemical support for models regarding the roles of checkpoint Rads as damage sensors in the DNA damage checkpoint response of human cells.
Lindsey-Boltz
L A
LA
Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, NC 27599, USA.
Bermudez
V P
VP
Hurwitz
J
J
Sancar
A
A
D000251:Adenosine Triphosphatases / Q000378:metabolism; D001665:Binding Sites; D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002460:Cell Line; D002474:Cell-Free System; D004247:DNA / Q000378:metabolism; D004249:DNA Damage; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D005092:Exonucleases / Q000302:isolation & purification / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D014176:Protein Biosynthesis; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D051818:Replication Protein C; D015398:Signal Transduction; D014158:Transcription, Genetic; D014162:Transfection
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D021122:Protein Subunits; C497564:RFC5 protein, human; C113575:Rad17 protein, human; D011994:Recombinant Proteins; C069930:rad9 protein; D004247:DNA; D005092:Exonucleases; C113574:Rad1 protein, human; D000251:Adenosine Triphosphatases; D051818:Replication Protein C