The Rad9-Hus1-Rad1 checkpoint clamp regulates interaction of TopBP1 with ATR.
TopBP1 serves as an activator of the ATR-ATRIP complex in response to the presence of incompletely replicated or damaged DNA. This process involves binding of ATR to the ATR-activating domain of TopBP1, which is located between BRCT domains VI and VII. TopBP1 displays increased binding to ATR-ATRIP in Xenopus egg extracts containing checkpoint-inducing DNA templates. We show that an N-terminal region of TopBP1 containing BRCT repeats I-II is essential for this checkpoint-stimulated binding of TopBP1 to ATR-ATRIP. The BRCT I-II region of TopBP1 also binds specifically to the Rad9-Hus1-Rad1 (9-1-1) complex in Xenopus egg extracts. This binding occurs via the C-terminal domain of Rad9 and depends upon phosphorylation of its Ser-373 residue. Egg extracts containing either a mutant of TopBP1 lacking the BRCT I-II repeats or a mutant of Rad9 with an alanine substitution at Ser-373 are defective in checkpoint regulation. Furthermore, an isolated C-terminal fragment from Rad9 is an effective inhibitor of checkpoint signaling in egg extracts. These findings suggest that interaction of the 9-1-1 complex with the BRCT I-II region of TopBP1 is necessary for binding of ATR-ATRIP to the ATR-activating domain of TopBP1 and the ensuing activation of ATR.
Lee
Joon
J
Division of Biology, California Institute of Technology, Pasadena, California 91125, USA.
Kumagai
Akiko
A
Dunphy
William G
WG
D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D064007:Ataxia Telangiectasia Mutated Proteins; D002352:Carrier Proteins / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000378:metabolism / Q000502:physiology*; D002868:Chromosomal Proteins, Non-Histone / Q000502:physiology*; D004268:DNA-Binding Proteins; D005786:Gene Expression Regulation; D008969:Molecular Sequence Data; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000502:physiology*; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000737:chemistry; D029867:Xenopus Proteins / Q000502:physiology*; D014982:Xenopus laevis / Q000378:metabolism*
D002352:Carrier Proteins; D018797:Cell Cycle Proteins; 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; D012694:Serine; C576898:Atr protein, Xenopus; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases; D000409:Alanine