Rad17 plays a central role in establishment of the interaction between TopBP1 and the Rad9-Hus1-Rad1 complex at stalled replication forks.
Rad17 is critical for the ATR-dependent activation of Chk1 during checkpoint responses. It is known that Rad17 loads the Rad9-Hus1-Rad1 (9-1-1) complex onto DNA. We show that Rad17 also mediates the interaction of 9-1-1 with the ATR-activating protein TopBP1 in Xenopus egg extracts. Studies with Rad17 mutants indicate that binding of ATP to Rad17 is essential for the association of 9-1-1 and TopBP1. Furthermore, hydrolysis of ATP by Rad17 is necessary for the loading of 9-1-1 onto DNA and the elevated, checkpoint-dependent accumulation of TopBP1 on chromatin. Significantly, a mutant 9-1-1 complex that cannot bind TopBP1 has a normal capacity to promote elevated accumulation of TopBP1 on chromatin. Taken together, we propose the following mechanism. First, Rad17 loads 9-1-1 onto DNA. Second, TopBP1 accumulates on chromatin in a manner that depends on both Rad17 and 9-1-1. Finally, 9-1-1 and TopBP1 dock in a Rad17-dependent manner before activation of Chk1.
Lee
Joon
J
Division of Biology 147-75, California Institute of Technology, Pasadena, CA 91125, USA.
Dunphy
William G
WG
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002352:Carrier Proteins; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D002843:Chromatin / Q000378:metabolism; D002868:Chromosomal Proteins, Non-Histone / Q000235:genetics / Q000378:metabolism*; D004261:DNA Replication; D004268:DNA-Binding Proteins; D029867:Xenopus Proteins / Q000235:genetics / Q000378:metabolism*; D014982:Xenopus laevis / Q000502:physiology
D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D002843:Chromatin; D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins; C511264:Hus1 protein, Xenopus; C511263:Rad1 protein, Xenopus; C509993:TopBP1 protein, Xenopus; D029867:Xenopus Proteins; C069930:rad9 protein; D000251:Adenosine Triphosphatases; C551927:Rad17 protein, Xenopus