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Papers In Press, published online ahead of print September 11, 2002
J. Biol. Chem, 10.1074/jbc.M207272200
Submitted on July 19, 2002
Revised on September 10, 2002
Accepted on September 11, 2002

Genotoxin-induced Rad9-Hus1-Rad1 (9-1-1) chromatin association is an early checkpoint signaling event

Pia Roos-Mattjus, Benjamin T. Vroman, Matthew A. Burtelow, Matthew Rauen, Alex K. Eapen, and Larry M. Karnitz

Oncology Dept., Mayo Clinic, Rochester, MN 55905

Corresponding Author: karnitz.larry{at}mayo.edu

Rad17, Rad1, Hus1, and Rad9 are key participants in checkpoint signaling pathways that block cell cycle progression in response to genotoxins. Biochemical and molecular modeling data predict that Rad9, Hus1, and Rad1 form a heterotrimeric complex, dubbed 9-1-1, which is loaded onto chromatin by a complex of Rad17 and the four small RFC subunits (Rad17-RFC) in response to DNA damage. It is unclear what checkpoint proteins or checkpoint signaling events regulate the association of the 9-1-1 complex with DNA. Here we show that genotoxin-induced chromatin binding of 9-1-1 does not require the Rad9 inducible phosphorylation site (Ser272). Although we found that Rad9 undergoes an additional phosphatidylinositol 3-kinase-related kinase (PIKK)-dependent post-translational modification, we also show that genotoxin-triggered 9-1-1 chromatin binding does not depend on the catalytic activity of the PIKKs ATM, ATR, or DNA-PK. Additionally, 9-1-1 chromatin binding does not require DNA replication, suggesting that the complex can be loaded onto DNA in response to DNA structures other than stalled DNA replication forks. Collectively, these studies demonstrate that 9-1-1 chromatin binding is a proximal event in the checkpoint signaling cascade.


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