Research Article

Cdc48 and a ubiquitin ligase drive disassembly of the CMG helicase at the end of DNA replication

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Science  24 Oct 2014:
Vol. 346, Issue 6208, 1253596
DOI: 10.1126/science.1253596

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Structured Abstract

Introduction

Chromosome replication is initiated by a universal mechanism in eukaryotic cells. This mechanism entails the assembly and activation at replication origins of the DNA helicase known as CMG (Cdc45-MCM-GINS), which is essential for the progression of replication forks. The replisome is built around the CMG helicase, which associates stably with DNA replication forks until the termination of DNA synthesis. The mechanism by which CMG is disassembled was unknown until now but is likely to represent a key regulated step at the end of chromosome replication.

Embedded Image

Regulated disassembly of the CMG helicase at the end of chromosome replication in budding yeast. It is very important that the CMG helicase is not displaced from replication forks during elongation, because it cannot be reloaded. When replication terminates, however, the ubiquitin ligase SCFDia2 and the Cdc48 segregase induce disassembly of the CMG helicase, leading to dissolution of the replisome.

Rationale

The CMG helicase exists only at DNA replication forks but can be isolated from extracts of S-phase budding yeast cells, after digestion of chromosomal DNA. We screened for posttranslational modifications of the CMG helicase that might regulate its function.

Results

Here we show that the CMG helicase is ubiquitylated during the final stages of chromosome replication in Saccharomyces cerevisiae, specifically on its Mcm7 subunit. The F-box protein Dia2 is essential in vivo for ubiquitylation of CMG, and the SCFDia2 ubiquitin ligase is also required to ubiquitylate CMG in vitro on its Mcm7 subunit in extracts of S-phase yeast cells. Ubiquitylated CMG exists only transiently in vivo, as it is rapidly disassembled in a reaction that is independent of the proteasome but requires the Cdc48/p97 segregase, which associates with ubiquitylated CMG. Consistent with these data, we show that Dia2 is essential for disassembly of the CMG helicase at the end of S phase in budding yeast. Rather than causing dissolution of the active helicase, Dia2 specifically induces the disassembly of terminated CMG complexes, which suggests that the helicase undergoes a change at the end of DNA replication, predisposing it for disassembly.

Conclusion

Our findings indicate that the end of chromosome replication in eukaryotes is controlled in a similarly complex fashion to the much-better-characterized initiation step. Our findings indicate that replisome disassembly is driven by the regulated dissolution of the stable CMG helicase, which uses its hexameric ring of Mcm2-7 proteins to encircle the parental DNA at replication forks. Our data identify two key features of helicase disassembly in budding yeast: First, there is an essential role for the F-box protein Dia2, which drives ubiquitylation of the CMG helicase on its Mcm7 subunit. Second, the Cdc48 segregase is required to break ubiquitylated CMG into its component parts. Once separated from GINS and Cdc45, the Mcm2-7 hexamer is less stable, so that all of the subunits of the CMG helicase are lost from the newly replicated DNA. Just as the main features of helicase assembly have been conserved across evolution from yeasts to humans, we envisage that disassembly of the CMG helicase will also involve a universal mechanism in eukaryotic cells, by which Cdc48/p97 drives disassembly of ubiquitylated CMG at the end of DNA replication.

How to stop after copying the genome

Replication is highly regulated: Failure to copy any part of the genome or copying parts of it more than once can cause genome instability with potentially disastrous consequences. Maric et al. and Priego Moreno et al. show that the DNA replication machinery, which stably encircles DNA during the duplication process, is actively disassembled once replication is complete (see the Perspective by Bell). The protein ring encircling the DNA is covalently modified, which allows it to be opened and the whole replication complex to be removed from DNA by a special disassembly complex.

Science, this issue 10.1126/science.1253596, p. 477; see also p. 418

Abstract

Chromosome replication is initiated by a universal mechanism in eukaryotic cells, involving the assembly and activation at replication origins of the CMG (Cdc45-MCM-GINS) DNA helicase, which is essential for the progression of replication forks. Disassembly of CMG is likely to be a key regulated step at the end of chromosome replication, but the mechanism was unknown until now. Here we show that the ubiquitin ligase known as SCFDia2 promotes ubiquitylation of CMG during the final stages of chromosome replication in Saccharomyces cerevisiae. The Cdc48/p97 segregase then associates with ubiquitylated CMG, leading rapidly to helicase disassembly. These findings indicate that the end of chromosome replication in eukaryotes is controlled in a similarly complex fashion to the much-better-characterized initiation step.

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