MOLECULAR FUNCTIONS OF THE RAD9A-RAD1-HUS1 DNA DAMAGE CHECKPOINT CLAMP IN GENOME MAINTENANCE AND TUMORIGENESIS
Patel, Darshil R
DNA damage checkpoint pathways are part of the broader cellular DNA damage response (DDR) that promotes genome maintenance when cells experience DNA damage. The RAD9A-HUS1-RAD1 (9-1-1) complex is a DDR component that functions as a heterotrimeric DNA binding clamp that promotes checkpoint signaling and DNA repair. Loss of 9-1-1 function has severe consequences, including embryonic lethality, genomic instability, subfertility and hypersensitivity to replication stress. To fully understand the role of the 9-1-1 complex in DNA repair, we first utilized a targeted mutation approach to identify functionally important residues of HUS1 that drive clamp formation, DNA interactions, and downstream effector functions. These studies revealed that both checkpoint signaling and DNA repair by the 9-1-1 complex separably promote genome maintenance. Next, a proteome-wide screen identified novel HUS1interactors and uncovered a new role for the 9-1-1 complex in regulating protein neddylation. As Hus1 loss results in radial chromosome formation and hypersensitivity to inter-strand crosslinking (ICL) agents, phenotypes also seen in the genome instability syndrome Fanconi Anemia (FA), we investigated the relationship between the 9-1-1 complex and the FA DNA repair pathway. Our studies suggested that the 9-1-1 complex was essential for recruitment of several FA proteins during ICL repair. Furthermore, the 9-1-1 complex also protected stalled replication forks against MRE11-dependent fork degradation. Overall, these data suggest a role for the 9-1-1 complex in coordinating multiple signaling and repair proteins required for accurate ICL repair. DDR proteins are known to protect against cancer-initiating mutations in normal cells; however they also support tumor growth at later stages, allowing cancers to tolerate elevated stress levels associated with malignant transformation. We explored the role of HUS1 in cancer, and discovered that Hus1 deficiency decreased cell transformation in cultured cells and decreased tumorigenesis in mouse models of lung and skin cancer. Expression analysis in multiple cancer cell lines revealed that HUS1 levels in cancer can increase through a mechanism involving alternative polyadenylation. In sum, this dissertation decodes distinct functions for the 9-1-1 complex in DNA repair and tumorigenesis, and identifies novel interactions between HUS1 and effectors important for genomic integrity and cell survival.
Biochemistry; Cellular biology; Molecular biology; Checkpoint; DNA Damage Response; DNA Repair; Genomic Stability; Replication Fork Stability; Tumorigenesis
Weiss, Robert S.
Schimenti, John C.; Smolka, Marcus B.
Biochemistry, Molecular and Cell Biology
Ph. D., Biochemistry, Molecular and Cell Biology
Doctor of Philosophy
dissertation or thesis