Mechanistic Functions Of The 9-1-1 Complex Subunit Hus1 In The Maintenance Of Genomic Integrity
Lim, Pei Xin
The mammalian RAD9A-HUS1-RAD1 (9-1-1) complex is a heterotrimeric clamp that promotes checkpoint signaling and DNA repair by functioning as a scaffold at DNA damage sites. While activation of the ATR checkpoint kinase via 9-1-1/TOPBP1 interactions is well established, less is known about checkpoint signaling-independent roles of 9-1-1. The 9-1-1 complex is thought to recruit DNA repair proteins from various repair pathways to damage sites via hydrophobic domains on the outer surface of the clamp. However, the molecular mechanisms and the physiological significance of these interactions remain mostly unknown. I hypothesize that 9-1-1 has a direct role in DNA repair that is coordinated with ATR checkpoint signaling to maintain genome integrity. This dissertation aims to elucidate the importance of checkpoint signaling-independent functions of HUS1 and the 9-1-1 complex using molecular and genetic approaches. First, we conducted the first comprehensive structure/function study of HUS1 by testing the ability of HUS1 mutants with various targeted mutations to complement the genotoxin sensitivity of Hus1-deficient fibroblasts. In this study, we elucidated functional residues of HUS1 that drive clamp assembly, DNA interactions, and downstream effector functions. Importantly, we found two hydrophobic pockets on the HUS1 outer surface that may serve as HUS1-effector interaction domains that are separable from the checkpoint activation function of 9-1-1. These results indicate that, once properly loaded onto damaged DNA, the 9-1-1 complex executes multiple, separable functions that promote genome maintenance. We also investigated the functional relationship between 9-1-1 complex and the homologous recombination repair (HRR) pathway by analyzing genetic interactions between Hus1 and Rad54. Previous studies suggested that Hus1 may play an important role in double strand break (DSB) repair, and Rad54-/- mice are mildly perturbed for HRR. We found that simultaneous Hus1 and Rad54 defects in mice and cultured cells caused synergistic effect on spontaneous and genotoxin-induced genomic instability, as well as male subfertility. These findings reveal Hus1 may directly participate in DSB repair pathways to respond to DNA damaging stresses. Together, these studies provide insights into novel functions of HUS1 that underlie a successful DNA damage response, which potentially could be exploited for targeted anticancer therapy.
RAD9-HUS1-RAD1; DNA damage response; checkpoint-repair coordination
Weiss, Robert S.
Alani, Eric; Schimenti, John C.
Ph. D., Genetics
Doctor of Philosophy
dissertation or thesis