Stabilisation of half MCM ring by Cdt1 during DNA insertion

Marina Guerrero-Puigdevall,Jordi Frigola,Narcis Fernandez-Fuentes

doi:10.1038/s41467-021-21932-8

Marina Guerrero-Puigdevall, Jordi Frigola + Show 1 more

Open Access

https://doi.org/10.1038/s41467-021-21932-8

Copy DOI

Abstract

Origin licensing ensures precise once per cell cycle replication in eukaryotic cells. The Origin Recognition Complex, Cdc6 and Cdt1 load Mcm2-7 helicase (MCM) into a double hexamer, bound around duplex DNA. The complex formed by ORC-Cdc6 bound to duplex DNA (OC) recruits the MCM-Cdt1 complex into the replication origins. Through the stacking of both complexes, the duplex DNA is inserted inside the helicase by an unknown mechanism. In this paper we show that the DNA insertion comes with a topological problem in the stacking of OC with MCM-Cdt1. Unless an essential, conserved C terminal winged helix domain (C-WHD) of Cdt1 is present, the MCM splits into two halves. The binding of this domain with the essential C-WHD of Mcm6, allows the latching between the MCM-Cdt1 and OC, through a conserved Orc5 AAA-lid interaction. Our work provides new insights into how DNA is inserted into the eukaryotic replicative helicase, through a series of synchronized events.

Highlights

Origin licensing ensures precise once per cell cycle replication in eukaryotic cells
Origin licensing starts with the origin recognition complex (ORC) binding to origin DNA in its ATP-bound form
An ORC-related member, Cdc[6], binds to the ORC in its own ATP-bound form. It is this ORC-Cdc[6] complex, bound to double-strand DNA and ATP, that further recruits the MCMs to proceed with the DH formation[3]

Summary

Introduction

Origin licensing ensures precise once per cell cycle replication in eukaryotic cells. The Origin Recognition Complex, Cdc[6] and Cdt[1] load Mcm[2,3,4,5,6,7] helicase (MCM) into a double hexamer, bound around duplex DNA. 1234567890():,; During origin licensing, eukaryotic cells load two MCMs into a head-to-head double hexamer around duplex DNA (DH)[1,2] This process, known as preRC assembly (from pre-replication complex), is the basis of chromosome replication regulation. The only preRC member that is not bound to DNA is Cdt[1], which forms a stable complex with MCM in solution, named MCM-Cdt[1] It is the MCM-Cdt[1] which, when recruited by the OC, will proceed with the helicase loading and lead to the DH formation

Methods

Results

Conclusion