Stabilisation of half MCM ring by Cdt1 during DNA insertion

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. During pre-Replication Complex, eukaryotic cells load two MCMs into a head-to-head Double Hexamer around duplex DNA (DH). Here the authors preRC assembly assay with purified proteins to reveal insights into S. cerevisiae's first steps that lead to the DH formation

We are grateful to P. Cherepanov and N. Cook for vectors and protocols (The Francis Crick Institute) and Jordi Torres for his assistance using the freezer mill (Institut de Recerca Biomedica de Lleida). N.F.-F. and J.F. are supported by MINECO (RYC-2015-17519 and RYC-2014-15837, BFU2016-79927-P, respectively). This work is also supported by Fundacio Bosch i Aymerich. M.G.-P. has been supported by private funding from Vanessa Nueda and collaborators of El crep de mi vida, Konig Restaurants and Rotary Club Banyoles