The SNM1B Nuclease Plays Important Roles in Maintaining Genome Stability.

Abstract

Efficient repair of damaged DNA is of critical importance to maintain genome integrity. Inherited mutations in repair genes result in genome instability disorders characterized by developmental defects and predisposition to cancer. Understanding the mechanisms underlying repair of DNA lesions will provide key insights into how genome stability is maintained as well as how defective repair leads to human disease. This dissertation focuses on elucidating the roles of the Snm1B nuclease in the repair of spontaneous and induced DNA damage. One of the most cytotoxic forms of DNA damage is the interstrand crosslink (ICL), which prevents strand separation and inhibits essential processes such as replication and transcription. ICL repair is complex and involves the coordination of many DNA repair pathways, including the Fanconia anemia (FA) pathway. FA is an inherited genome instability disorder characterized by cellular sensitivity to ICLs, developmental defects and cancer predisposition. There are 13 FA complementation groups; however, the functions of the FA factors as well as the mechanism of ICL repair remain poorly understood. The studies described in this thesis provide insights into the regulation of cellular responses to ICLs by Snm1B. Previous studies suggest Snm1B promotes replication fork collapse. I demonstrate that Snm1B is critical for activation of ATR, the central ICL signal transduction protein kinase. I establish that Snm1B depletion reduces phosphorylation of ATR and demonstrate that Snm1B functions epistatically to the FA factor, FANCD2 and promotes efficient FANCD2 localization. These studies indicate that Snm1B has key roles in checkpoint signaling as well as in processing of the ICL. In addition, I demonstrate that Snm1B functions in the response to replication stress downstream of ATR activation. This work has established important roles for Snm1B in protecting the genome from spontaneous and induced DNA damage during replication. The evidence presented here has identified multiple roles for Snm1B in DNA repair processes at the replication fork, both upstream and downstream of the ATR kinase. Together, these findings provide evidence that Snm1B is critical for ATR-mediated signaling and the FA pathway, and suggest that mutations in Snm1B may underlie human genome instability diseases such as Fanconi anemia.