PROJECT SUMMARY Genomic DNA is constantly challenged by DNA damage either spontaneously induced during cellular metabolism or generated by exogenous DNA damaging agents. During DNA replication, DNA lesions can cause the stalling or collapse of replication forks. Fork collapse results in the formation of DNA double-strand breaks (DSBs). MCM8 and MCM9 (MCM8-9) form a helicase complex that promotes the repair of DSBs by homologous recombination. We recently identified MCM8IP as a novel interactor of MCM8-9 that maintains genomic integrity after replication stress. In particular, we showed that MCM8IP promotes DSB repair by homologous recombination, facilitates the restart of replication forks arrested by DNA lesions and protects cells from DNA damage generated by replication stress-inducing agents. Despite these important preliminary findings, the precise mechanisms of action exhibited by MCM8IP, MCM8 and MCM9 during DNA recombination and replication remain to be elucidated. MCM8 or MCM9 have been reported to act as tumor suppressors and recent cancer genomic analyses have evidenced mutations in MCM8, MCM9 and MCM8IP in multiple cancer types. The goals of this proposal are to define the precise biochemical and cellular activities displayed by the MCM8IP-MCM8-9 complex for suppressing genomic instability and to examine how these activities are affected by mutations in MCM8IP, MCM8 and MCM9 identified in tumors. In particular, we propose 1) to define the biochemical activities and physical interactions exhibited by the MCM8IP-MCM8-9 complex for preserving genomic integrity; 2) to elucidate the mechanisms by which MCM8IP-MCM8-9 promotes DSB repair and ensures replication fork progression in response to DNA damage; 3) to evaluate the contribution of MCM8IP, MCM8 and MCM9 cancer-associated mutations to genomic instability. Our approach will utilize innovative proteomic methods, state-of-the-art genome editing technologies, single-molecule analyses of replication dynamics, electron microscopy and super-resolution imaging. We anticipate that our studies will define the unique mechanisms employed by the MCM8IP-MCM8-9 complex to suppress genomic instability and will provide insights into the potential contribution of MCM8IP, MCM8 and MCM9 cancer- associated mutations to cancer etiology.