Analysis of a DNA Damage-Inducible Checkpoint Complex

Project Summary: The integrity of our genomes is threatened continuously by DNA damage caused by errors generated during replication, metabolically produced reactive oxygen species, spontaneous depurination, ultraviolet and ionizing radiation, and exogenous agents. This DNA damage triggers two interdependent cellular responses: 1) the mobilization of specific DNA repair machinery, which acts to correct the lesion, and 2) the activation of checkpoint signaling pathways, which induce cell cycle arrest and regulate DNA repair. A key node in the regulation of both responses is the Rad9-Hus1-Rad1 (911) complex. This heterotrimeric clamp-like complex is loaded onto DNA at sites of damage, where it activates the ATR-Chk1 checkpoint signaling pathway. Additionally, in vitro studies show that the 911 complex directly interacts with and stimulates the Base Excision Repair (BER) machinery. Collectively, these observations indicate that the 911 complex serves as sliding clamp that orchestrates and integrates checkpoint activation and DNA repair to promote genome stability. Despite this progress, critical questions remain. First, it is not clear how the 911 complex participates in BER in intact cells. Second, even though Rad9 is highly phosphorylated, an event important for Rad9-mediated checkpoint activation, it is not known what kinases phosphorylate Rad9 or how they regulate Rad9 function. Here we propose three specific aims that will determine how the 911 complex participates in BER in cells, and will examine how two cell cycle-regulated kinases that bind and phosphorylate Rad9 regulate the function of Rad9. These studies will combine biochemical, genetic, and cell biology approaches to decipher the molecular mechanisms whereby the 911 clamp regulates these cellular responses to DNA damage. Thus, these studies will further our understanding of a complex that plays key roles in maintaining genomic stability and preventing cancer.