MCM Double Hexamer Loading Visualised with Human Proteins

Eukaryotic DNA replication begins with the loading of the MCM replicative DNA helicase as a head-to-head double hexamer (DH) at origins of DNA replication ^1–3 . Our current understanding of how DH is assembled by the Origin Recognition Complex (ORC), CDC6 and CDT1 comes mostly from budding yeast. Here we characterise human DH (hDH) loading using biochemical reconstitution and cryo-electron microscopy with purified proteins. We show that hDH engages DNA differently from yeast (yDH), and generates ∼5 base pairs of unwound DNA at the interface between hexamers, as seen in hDH isolated from cells ⁴ . We identify several differences from yeast in the order of factor recruitment and dependencies during hDH assembly. Unlike yeast ^5–8 , the ORC6 subunit of ORC is not essential for initial MCM recruitment or hDH loading, but contributes to an alternative hDH assembly pathway requiring an intrinsically disordered region (IDR) in ORC1, which may work through a novel MCM-ORC (hMO*) intermediate. Our work presents a detailed view of how DHs are assembled in an organism utilising sequence-independent replication origins, it provides further evidence for diversity in eukaryotic DH assembly mechanisms ⁹ , and it represents the first step toward reconstitution of DNA replication initiation with purified human proteins.