The ORC ubiquitin ligase OBI1 promotes DNA replication origin firing

Philippe Coulombe,Slavica Stanojcic,Marcel Méchali,Axel Delamarre,Stéphane Bocquet,Yvon Sterkers,Isabelle Peiffer,Joelle Nassar

doi:10.1038/s41467-019-10321-x

Abstract

DNA replication initiation is a two-step process. During the G1-phase of the cell cycle, the ORC complex, CDC6, CDT1, and MCM2–7 assemble at replication origins, forming pre-replicative complexes (pre-RCs). In S-phase, kinase activities allow fork establishment through (CDC45/MCM2–7/GINS) CMG-complex formation. However, only a subset of all potential origins becomes activated, through a poorly understood selection mechanism. Here we analyse the pre-RC proteomic interactome in human cells and find C13ORF7/RNF219 (hereafter called OBI1, for ORC-ubiquitin-ligase-1) associated with the ORC complex. OBI1 silencing result in defective origin firing, as shown by reduced CMG formation, without affecting pre-RC establishment. OBI1 catalyses the multi-mono-ubiquitylation of a subset of chromatin-bound ORC3 and ORC5 during S-phase. Importantly, expression of non-ubiquitylable ORC3/5 mutants impairs origin firing, demonstrating their relevance as OBI1 substrates for origin firing. Our results identify a ubiquitin signalling pathway involved in origin activation and provide a candidate protein for selecting the origins to be fired.

Highlights

DNA replication initiation is a two-step process
After ORC binding, CDC6 and CDT1 assemble at origins and permit the licensing reaction that culminates with the loading onto chromatin of the replicative helicase MCM2/7 in its inactive state, defining the pre-replicative complexes
We focused on C13ORF7/RNF219, an uncharacterised potential E3 ubiquitin ligase that we called OBI1 (ORC ubiquitin-ligase-1)

Summary

Introduction

DNA replication initiation is a two-step process. During the G1-phase of the cell cycle, the ORC complex, CDC6, CDT1, and MCM2–7 assemble at replication origins, forming prereplicative complexes (pre-RCs). Potential DNA replication origin sites (hereafter called origins) are established during the G1 phase of the cell cycle through a highly regulated process known as replication licensing[3,4] (for review). After ORC binding, CDC6 and CDT1 assemble at origins and permit the licensing reaction that culminates with the loading onto chromatin of the replicative helicase MCM2/7 in its inactive state, defining the pre-replicative complexes (preRCs). As cells enter S phase, the MCM2/7 helicase is activated at preRCs through CDK (Cyclin-dependent kinase) and DDK (DBF4dependent kinase) activities[6] This leads to the association of CDC45 and the GINS complex to chromatin-bound MCM2/7 and, to the formation of the active CMG complex[7]. The properties of OBI1 suggest that it could be a replication origin selector essential for DNA replication origin activation during S phase

Methods

Results

Conclusion