MCM8/9 unwinding activation, specificity, and stalled fork remodeling - Project Summary – Both endogenous and exogenous agents present continuous challenges to faithful DNA replication and genomic maintenance of a species. During S-phase, the DNA duplex is exposed as single- strands, and the genome becomes most susceptible to deleterious single- and double-strand breaks that can become molecular hallmarks of cancers. Fortunately, there are several pathways and processes to stabilize and restart replication forks should they stall, but defects at various stages of replication fork remodeling are linked to several genetic diseases with increased susceptibilities to a multitude of genomic stressors leading to cancer, cellular aging, intellectual disabilities linked to aneuploidy, and infertility. Mutation in two recently identified genes, MCM8 and MCM9, have been associated with defects in both mitotic and meiotic replication/ recombination, resulting in pathogenic genomic intermediates in mammals. Results from knockout mice, genetic mutations, phenotypes in humans, and functional cellular studies are informative but have only scratched the surface of our mechanistic understanding, as specific roles or activities for MCM8/9 have not been delineated from other HR helicases. MCM8/9 is unique, structured as an alternating trimer of dimers heterohexameric complex, with specific motifs and domains that can direct interactions. We hypothesize that MCM8/9 intersects the Fanconi anemia protein fork stabilizing pathway by directly interacting with FANCD2I to mediate the transition of stalled forks into stabilized forks through facilitated RAD51 deposition. A suite of preliminary cryo-EM structures will allow us to propose a mechanism of unwinding activation for MCM8/9 that includes a significant rotation and tier compaction upon binding of the interacting protein, HROB, and DNA. To test this, we will determine the structure-function relationships for MCM8/9/HROB for several DNA transactions involving the MCM9-interacting recombinase, RAD51. Our preliminary data shows a clear interaction of MCM8/9 with FANCD2I, intersecting with the Fanconi anemia fork stalled fork protection pathway. We will determine how unique motifs and domains within MCM8 and MCM9 direct specificity of repair processes through discrete interactions. Finally, we will work to understand how MCM8/9 performs the early stages of fork remodeling to deposit RAD51 for stabilization using advanced biochemical reconstitutions and biophysical monitoring. The proposed research program is highly integrated and interdisciplinary using advanced cryoEM structure determination, biochemical reconstitution of fork remodeling processes, and cellular DNA damage response assays to better understand MCM8/9 functions both in vitro and in cells. Results from this proposal will provide a clearer understanding of the specificity of the MCM8/9/HROB complex within the context of many other known DNA repair factors. This will provide insight into the structure function activities and interactions of MCM8/9 that work to protect stalled forks and provide insight into genomic instability, cancer development, and infertility caused by patient mutations in these genes.
