MCM8/9 and FANCD2 interact within a shared pathway in response to replication stress caused by DNA crosslinks.

The Fanconi anemia (FA) protein FANCD2, and MCM8/9 heterohexameric helicase complex are critical for maintaining genomic integrity in response to replication stress, however, the nature of their relationship remains unclear. Here, we show that MCM8/9 interacts and functionally cooperates with FANCD2 within a complex during the repair of DNA interstrand crosslinks (ICLs). Using immunofluorescence and co-immunoprecipitation studies, we show that MCM8/9 interacts with the FANCD2 complex through its core domain. FANCD2 is essential for the recruitment of MCM8/9 to ICL damage induced nuclear foci but acts independently of FANCD2 monoubiquitination. Although MCM8/9 foci formation requires its intact ATPase activity, the BRCv motif within the MCM9 C-terminal extension (CTE) and the accessory protein, HROB, these are not required for FANCD2 binding, highlighting a distinction between physical interaction and functional activation. Interestingly, FANCD2 foci formation increases in MCM8 or MCM9 knockout cells or with knockdown of the activator, HROB, suggesting that MCM8/9 functions to mitigate replication-associated stress. γH2AX DNA damage assays and cell survival assays show that combined loss of MCM9 and FANCD2 do not cause additive DNA damage beyond individual knockouts, indicating an epistatic relationship of MCM8/9 with FANCD2, functioning in the same DNA repair pathway. Together, our findings identify MCM8/9 as a downstream interactor and effector of FANCD2/I critical for resolving ICL induced DNA damage.