GSE202066
The Human Pre-replication Complex is an Open Complex
In eukaryotes, DNA replication initiation requires assembly and activation of the minichromosome maintenance (MCM) 2-7 double hexamer (DH) to melt origin DNA strands. However, the mechanism for this initial melting is unknown. Here, we report a 2.59-Å cryo-electron microscopy structure of the endogenously purified human MCM-DH (hMCM-DH), also known as the pre-replication complex. In this structure, the hMCM-DH shows a constricted central channel with a diameter of only 13 Å at the hexamer interface. This unusual conformation untwists and stretches the DNA strands such that almost a half turn of the bound duplex DNA is distorted with one-base pair completely separated, generating an initial open structure (IOS) at the hexamer junction. This IOS is captured and stabilized by zinc fingers (ZFs) of MCM2 and MCM5 from opposing hexamers. Disturbing the interaction between MCM5-ZF and the IOS demonstrably inhibits DH formation and replication initiation. High-resolution mapping of hMCM-DH footprints indicates that IOSs are distributed across the human genome in large clusters aligning perfectly with initiation zones to safeguard robust stochastic origin firing. This work unravels an intrinsic mechanism that couples DH formation with initial DNA melting to license replication initiation in human cells. Overall design: Mapping of the hMCM-DH footprints in human cells