Investigations at the Stalled Fork: Potential Pathways Involving the MCM8/9 Complex in Fork Stability




Klinzing, Kathleen

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Efficiency of DNA replication is crucial to cell survival. Stalled forks must be resolved quickly to prevent fork collapse resulting in double-strand breaks (DSBs). The MCM8/9 helicase is hypothesized to play a role in both protection of DNA at stalled forks and in repair of DSBs. Mutations in MCM8/9 have been attributed to cancer and infertility. MCM9 has a unique C-terminal extension (CTE) of undetermined function that contains a BRCv motif thought to be important in mediating interactions with Rad51 during homologous recombination following DSBs. MCM9 truncations containing the CTE as well as amino acid substitutions within the BRCv were created and purified to determine the specificity of the CTE with Rad51. A GST pulldown with Rad51 confirmed a weak interaction between the CTE of MCM9 and Rad51. To evaluate the role of MCM8/9 at stalled forks, a CRISPR-Cas9 generated MCM9 knockout cell line was used to assess MCM8/9-mediated protection of stalled or reversed forks from nucleolytic degradation using DNA fiber assays. Interestingly, fork stability was restored in MCM9 knockout cells when BRCA1 was also knocked down with siRNA. These results suggest that MCM8/9 plays a non-redundant role to BRCA1 in maintenance of replication fork integrity and protection of stalled replication forks. Understanding the proteins that function at stalled replication forks and the order in which they arrive could provide avenues for development of therapeutic targets.



DNA repair., Homologous recombination., HU., MCM9.