Abstract 1772 Multiple pathways for licensing human replication origins

The loading of replicative helicases constitutes an obligatory step in the assembly of DNA replication machineries and in constructing bidirectional replication forks. In eukaryotes, the replicative helicase motor is the Mcm2-7 hexamer that is deposited onto DNA by the origin recognition complex (ORC) and co-loader proteins as a head-to-head MCM double hexamer to license origins. Much of our knowledge of eukaryotic origin licensing has been derived from studies of the S. cerevisiae model system. By contrast, the mechanisms of origin licensing in higher eukaryotes remain poorly understood. Here, we use biochemical reconstitution and electron microscopy (EM) to reconstruct the MCM loading pathway in the human system. Unexpectedly, we find that – contrary to S. cerevisiae – ORC's Orc6 subunit is not required for human MCM loading, although it can stimulate this event in certain conditions. EM analyses identify several intermediates on route to MCM double hexamer formation in the presence and absence of Orc6, including an abundant closed-ring single MCM hexamer intermediate loaded on DNA. Cryo-EM structures of Mcm2-7 before and after loading reveal a propensity of human Mcm2-7 hexamers to dimerize without the help of other loading factors. Collectively, our findings suggest that human replication origins are licensed through two different pathways, an Orc6-dependent one and an Orc6-independent one, and that double hexamer formation can proceed through dimerization of two independently loaded single Mcm2-7 hexamers. This redundancy in human MCM loading pathways likely provides resilience under cellular conditions and ensures that a sufficient number of origins are licensed for efficient DNA replication. This work was supported by the National Institutes of Health grants 5R01GM141313 to F.B., the NIH predoctoral program in Biophysics T32-GM008283 (support for O.H.), and F31CA278331 to O.H.