Multiple pathways for licensing human replication origins.

The loading of replicative helicases constitutes an obligatory step in the assembly of DNA replication machineries. In eukaryotes, the MCM2-7 replicative helicase motor is deposited onto DNA by the origin recognition complex (ORC) and co-loader proteins as a head-to-head MCM double hexamer to license replication origins. Although extensively studied in the budding yeast model system, the mechanisms of origin licensing in higher eukaryotes remain poorly defined. Here, we use biochemical reconstitution and electron microscopy (EM) to reconstruct the human MCM loading pathway. Unexpectedly, we find that, unlike in yeast, ORC's Orc6 subunit is not essential for human MCM loading but can enhance loading efficiency. EM analyses identify several intermediates en route to MCM double hexamer formation in the presence and absence of Orc6, including an abundant DNA-loaded, closed-ring single MCM hexamer intermediate that can mature into a head-to-head double hexamer through different pathways. In an Orc6-facilitated pathway, ORC and a second MCM2-7 hexamer are recruited to the dimerization interface of the first hexamer through an MCM-ORC intermediate that is architecturally distinct from an analogous intermediate in yeast. In an alternative, Orc6-independent pathway, MCM double hexamer formation proceeds through dimerization of two independently loaded single MCM2-7 hexamers, promoted by a propensity of human MCM2-7 hexamers to dimerize without the help of other loading factors. This redundancy in human MCM loading pathways likely provides resilience against replication stress under cellular conditions by ensuring that enough origins are licensed for efficient DNA replication. Additionally, the biochemical reconstitution of human origin licensing paves the way to address many outstanding questions regarding DNA replication initiation and replication-coupled events in higher eukaryotes in the future.