Reconstitution of human CMG helicase ubiquitylation by CUL2LRR1 and multiple E2 enzymes.

Thanh Thi Le,Johanna Ainsworth,Cristian Polo Rivera,Thomas Macartney,Karim P M Labib

doi:10.1042/bcj20210315

Thanh Thi Le, Johanna Ainsworth + Show 3 more

Open Access

https://doi.org/10.1042/bcj20210315

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Abstract

Cullin ubiquitin ligases drive replisome disassembly during DNA replication termination. In worm, frog and mouse cells, CUL2LRR1 is required to ubiquitylate the MCM7 subunit of the CMG helicase. Here, we show that cullin ligases also drive CMG-MCM7 ubiquitylation in human cells, thereby making the helicase into a substrate for the p97 unfoldase. Using purified human proteins, including a panel of E2 ubiquitin-conjugating enzymes, we have reconstituted CMG helicase ubiquitylation, dependent upon neddylated CUL2LRR1. The reaction is highly specific to CMG-MCM7 and requires the LRR1 substrate targeting subunit, since replacement of LRR1 with the alternative CUL2 adaptor VHL switches ubiquitylation from CMG-MCM7 to HIF1. CUL2LRR1 firstly drives monoubiquitylation of CMG-MCM7 by the UBE2D class of E2 enzymes. Subsequently, CUL2LRR1 activates UBE2R1/R2 or UBE2G1/G2 to extend a single K48-linked ubiquitin chain on CMG-MCM7. Thereby, CUL2LRR1 converts CMG into a substrate for p97, which disassembles the ubiquitylated helicase during DNA replication termination.

Highlights

Eukaryotic cells synthesise a single copy of their chromosomes in each cell cycle [1–3]
The catalytic core of the CMG helicase comprises a hexameric ring of the six MCM2–7 ATPases [19], which is stabilised by association with the CDC45 protein and the GINS complex and encircles the template for leading strand DNA synthesis at replication forks [20]
To isolate the CMG helicase from proliferating cells, we used CRISPR–Cas9 to modify both alleles of the SLD5 gene in human RPE1 cells, in order to introduce green fluorescent protein (GFP) at the amino terminus of the SLD5 subunit of GINS (Supplementary Figure S1)

Summary

Introduction

Eukaryotic cells synthesise a single copy of their chromosomes in each cell cycle [1–3]. The control of chromosome duplication is achieved by the regulated assembly and disassembly of the CMG DNA helicase (CMG = CDC45_MCM2-7_GINS), which associates with a range of other factors at replication forks to form the eukaryotic replisome [4–9]. We explore the regulation of CMG ubiquitylation in human cells and report the in vitro reconstitution of human CMG-MCM7 ubiquitylation, involving neddylated CUL2LRR1 and multiple ubiquitin-conjugating enzymes. These findings confirm CMG-MCM7 as the first nuclear substrate of mammalian CUL2LRR1, misregulation of which has been linked to several human cancers [39–42]

Results and discussion

Materials and methods