Recruitment of Mcm10 to Sites of Replication Initiation Requires Direct Binding to the MCM Complex [DNA and Chromosomes]

December 30th, 2015 by Douglas, M. E., Diffley, J. F. X.

Mcm10 is required for the initiation of eukaryotic DNA replication and contributes in some unknown way to the activation of the Cdc45-MCM-GINS (CMG) helicase. How Mcm10 is localised to sites of replication initiation is unclear, as current models implicate direct binding to MCM to play a role, but the details and functional importance of this interaction have not been determined. Here, we show that purified Mcm10 can bind both DNA-bound double hexamers and soluble single hexamers of MCM. The binding of Mcm10 to MCM requires the Mcm10 C-terminus. Moreover the binding site for Mcm10 on MCM includes the Mcm2 and Mcm6 subunits, and overlaps that for the loading factor Cdt1. Whether Mcm10 recruitment to replication origins depends on CMG helicase assembly has been unclear. We show that Mcm10 recruitment occurs via two modes: low affinity recruitment in the absence of CMG assembly (G1-like), and high affinity recruitment when CMG assembly takes place (S-phase-like). Mcm10 that cannot bind directly to MCM is defective in both modes of recruitment, and unable to support DNA replication. These findings indicate that Mcm10 is localised to replication initiation sites by directly binding MCM through the Mcm10 C-terminus.