Roles of the SNM1B DNA Nuclease in Resolution of Replication Stress and Maintenance of Genome Stability.

Abstract

Genomic DNA is damaged through exposure to exogenous and endogenous agents as well as during cellular processes such as DNA replication. Defects in cellular responses to DNA damage can lead to an accumulation of unrepaired or misrepaired lesions and ultimately, increased genome instability. DNA replication ensures the accurate transmission of the information encoded in the genome to daughter cells. Progression of DNA replication can be impaired or blocked, which leads to replication fork stalling. If stalled forks are not properly restarted, they can collapse, resulting in chromosomal breaks, deletions, and translocations. Therefore, replication-associated DNA damage has been hypothesized as one important source of genome instability associated with cancer initiation and progression. Furthermore, mutations in DNA repair genes result in inherited genome instability disorders characterized by developmental defects and cancer predisposition. Uncovering the cellular mechanisms that repair DNA damage is critical for understanding how cells maintain genome stability and thereby prevent deleterious human diseases. DNA nucleases play a key role in resolving stalled and collapsed replication forks, but the molecular events involved in these processes are not fully defined. This dissertation addresses how the DNA nuclease SNM1B plays critical roles in preventing replication-associated DNA damage. I demonstrate that SNM1B is not required for the initial detection of a stalled replication fork or for initiating early signaling events. I show that SNM1B is instrumental in stabilizing stalled replication forks by nucleolytically processing aberrant DNA structures at stalled and collapsed forks which allows for the recruitment of key DNA repair factors, FANCD2, BRCA1, and Rad51. Furthermore, I found that SNM1B plays a key role in preventing stalled and collapsed replication forks in unperturbed cells suggesting it is critical in responding to replication-associated DNA damage that occurs spontaneously during cellular proliferation. I also identified a residue within SNM1B that is critical for its localization to sites of stalled forks and established that SNM1B protein levels are modulated in the cell. Altogether, these findings illustrate that SNM1B has critical functions during the repair of replication-associated DNA damage, thereby ensuring successful replication of the genome and preventing potentially deleterious chromosomal aberrations.