Mechanism of head-to-head MCM double-hexamer formation revealed by cryo-EM.

Thomas C R Miller,Julia F Greiwe,John F X Diffley,Julia Locke,Alessandro Costa

doi:10.1038/s41586-019-1768-0

Thomas C R Miller, Julia F Greiwe + Show 3 more

Open Access

https://doi.org/10.1038/s41586-019-1768-0

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Journal: NatureJobs	Publication Date: Nov 20, 2019
Citations: 119	License type: implied-oa

Affiliation: The Francis Crick Institute

Abstract

In preparation for bidirectional replication, the origin recognition complex (ORC) loads two MCM helicases forming a head-to-head double hexamer (DH) around DNA1,2. How DH formation occurs is debated. Single-molecule experiments suggest a sequential mechanism whereby ORC-dependent loading of the first hexamer drives second hexamer recruitment3. In contrast, biochemical data show that two rings are loaded independently via the same ORC-mediated mechanism, at two inverted DNA sites4,5. We visualized MCM loading using time-resolved EM, to identify DH formation intermediates. We confirm that both hexamers are recruited via the same interaction between the MCM and ORC C-terminal domains, and identify the mechanism for coupled MCM loading. A first loaded hexamer locked around DNA is recognized by ORC, which unexpectedly engages the N-terminal homo-dimerization interface of MCM. In this configuration, ORC is poised to direct second hexamer recruitment in an inverted orientation, suitable for DH formation. Our data reconcile two apparently contrasting models.

Highlights

Genome replication in eukaryotes is tightly controlled to ensure that chromosomes are copied only once per cell cycle
double hexamer (DH) mark origin DNA that can support replisome assembly and, once activated, the MCMs unwind DNA providing the template for replicative polymerases to perform bidirectional replication
Bead-free helicase loading reactions analysed by electron microscopy (EM) show efficient DH formation as well as class averages containing isolated MCM-Cdt[1], origin recognition complex (ORC), nucleosome or nucleosome close to ORC (Fig. 1d and Extended Data Fig. 1b)

Summary

Introduction

Genome replication in eukaryotes is tightly controlled to ensure that chromosomes are copied only once per cell cycle. Combined with the observation that two inverted ORC binding sites promote efficient MCM loading[5], these data support a model whereby two distinct ORC-DNA engagement events symmetrically load two MCM rings via the same mechanism. Bead-free helicase loading reactions analysed by EM show efficient DH formation as well as class averages containing isolated MCM-Cdt[1], ORC, nucleosome or nucleosome close to ORC (Fig. 1d and Extended Data Fig. 1b).

Results

Conclusion