Structural Activation of DNA Unwinding by MCM8/9/HROB

The minichromosomal maintenance MCM8 and MCM9 proteins form a heterohexameric complex that acts to unwind or remodel duplex DNA in DNA recombination and repair pathways. Mutations or absence of MCM8/9 have been linked to infertility, sex-specific deficiencies, and several cancers. Recently, HROB has been identified as a critical cofactor of MCM8/9; however, the mechanism underlying activation of MCM8/9 DNA binding and unwinding remain unclear. Here, we present dynamic structures of MCM8/9 with DNA, HROB and ATP analogs using cryo-electron microscopy. DNA binding induces a pronounced rotational rearrangement between the N-terminal DNA binding and C-terminal AAA⁺ ATPase domains, reorganizing DNA-binding loops into a staircase configuration that supports DNA engagement. Remarkably, HROB associates with both halves of the heterohexamer and drives a similar rotation prior DNA binding for localizing MCM8/9 to sites of crosslink damage and unwinding, culminating in a unified mechanistic model for MCM8/9 helicase function and its activation by HROB.