HUMAN MUTS HOMOLOGUES hMSH4 AND hMSH5: FROM PROTEIN DEGRADATION TO DNA DAMAGE RESPONSE AND REPAIR

Thesis (Ph.D.), Washington State UniversityDNA double-strand breaks (DSBs) are among the most deleterious forms of DNA damage and the leading causes of genomic instability, cell death and cancer. Cells have evolved a complicated network of DNA damage response (DDR) pathways to deal with DSBs and to maintain genomic stability. The DDR network must be sensitive and effective, but more importantly, well controlled via a myriad of post-translational modifications of key DDR factors. Ubiquitination and protein degradation play an essential role here, not only in the negative control of the DDR signaling, but also in the facilitation of protein accumulation at DSB sites. This dissertation aims to explore the independent functions of human MutS homologues hMSH4 and hMSH5 in mitotic cells. Previous work has shown that the MSH4-MSH5 complex participates in meiotic homologous recombination (HR) and crossover formation. Here, we hypothesize that the protein degradation of hMSH4 is critical for promoting non-homologous end-joining (NHEJ)-mediated DSB repair. Our lab has characterized the interaction between hMSH4 and the von Hippel-Lindau (VHL) binding protein 1 (VBP1). We address that VBP1 regulates the homeostasis of hMSH4 through both proteasome and autophagy pathways. Specifically, we identify p97 as a new binding partner of VBP1, and the VBP1-VHL-p97 complex targets hMSH4 for ubiquitination and degradation. The crossover recombination is essential for meiosis to separate homologous chromosomes, but is detrimental to mitotic cells because of potential chromosomal translocations. To explore the hMSH4-independent functions of hMSH5, we identify FANCJ as a novel and direct binding partner of hMSH5. We demonstrate that hMSH5 promotes cell survival in response to camptothecin (CPT) treatment. Cells deficient in hMSH5 show compromised HR, aberrant activation of Chk1 and abnormal cell cycle progressions after CPT treatment. These defects are likely caused by a reduced, hMSH5-dependent recruitment of FANCJ to the chromatin. Furthermore, loss of FANCJ rescues the sensitivity to CPT in hMSH5- depleted cells. Together, we demonstrate the existence of a physical and functional interplay between hMSH5 and FANCJ in mediating replication stress and DSB repair. And we conclude that the protein degradation of hMSH4 dissociates the hMSH4-hMSH5 complex to promote NHEJ and hMSH5-dependent replication-associated DSB repair.Program in Molecular Biosciences, Washington State Universit