Regulation of ATR Signaling by CINP and RPA

Abstract

Genetic instability is a hallmark of cancer. The ATR-ATRIP complex functions at the apex of a cell cycle checkpoint signaling pathway that is critical during every cell division cycle to maintain genome integrity. ATR activity is critical for regulating the firing of replication origins, stabilizing and repairing damaged replication forks, and preventing the premature onset of mitosis. I have developed a model system for identifying checkpoint protein localization to stalled replication forks using CHIP. By stalling replication fork at a defined site on an episome, I have created a system with greater resolution than that conventionally used for protein localization studies. In collaboration with additional lab members, I have also identified a novel ATR signaling regulator, CINP. CINP interacts with ATR-ATRIP and is required for ATR-dependent Chk1 phosphorylation and maintenance of the G2 checkpoint. Finally, I have also examined the recruitment of checkpoint proteins to sites of DNA damage. This recruitment of ATR-ATRIP is mediated by an interaction between the checkpoint recruitment domain (CRD) of ATRIP and the ssDNA binding protein RPA. I found that two other checkpoint proteins, Rad9 and Mre11, contain a similar acid sequence, and that the CRD domains of ATRIP, Rad9, and Mre11, all contact the basic cleft of RPA70N in a similar manner. The ability of Rad9 to relocalize to DNA damage sites is compromised when the RAD9-RPA70 interaction is disrupted. Furthermore, mutations in the Rad9 CRD domain cause hypersensitivity to DNA damage and compromise ATR-dependent CHK1 phosphorylation. Mutations within the RPA70N OB fold impair checkpoint activation, but do not interfere with the DNA replication. My research has resulted in the development of a system for identifying checkpoint proteins recruited to stalled replication forks, identified a novel regulator of ATR signaling, and identified a protein-protein interaction surface within RPA70 that makes contacts with multiple checkpoint proteins to promote ATR signaling.