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CRL2LRR1 is required for CMG unloading during vertebrate replication termination

ABSTRACT: A key event during eukaryotic replication termination is the removal of the CMG helicase from chromatin. CMG unloading involves ubiquitylation of its MCM7 subunit and the action of the p97 ATPase. Using a proteomic screen in Xenopus egg extracts, we identified factors that are retained on chromatin when CMG unloading is blocked and identified the E3 ubiquitin ligase CRL2LRR1 and several other potential regulators of termination including a specific p97 complex. We show that CRL2LRR1 recruitment coincides with MCM7 ubiquitylation and occurs only when replisomes converge. In the absence of CRL2LRR1, MCM7 is not ubiquitylated, CMG unloading is inhibited, and many replisome components including DNA pol are retained on DNA. Our data identify CRL2LRR1 as a master regulator of replisome disassembly during vertebrate DNA replication termination.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Xenopus Laevis (african Clawed Frog)

TISSUE(S): Egg

SUBMITTER: Mario Oroshi

LAB HEAD: Markus Räschle

PROVIDER: PXD004828 | Pride | 2017-02-02

REPOSITORIES: Pride

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			Action	DRS
	20160424_QEp7_MaRa_SA_mr4p76_1A.raw	Raw
	20160424_QEp7_MaRa_SA_mr4p76_1B.raw	Raw
	20160424_QEp7_MaRa_SA_mr4p76_1C.raw	Raw
	20160424_QEp7_MaRa_SA_mr4p76_1D.raw	Raw
	20160424_QEp7_MaRa_SA_mr4p76_2A.raw	Raw

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Immunoprecipitation of p97 from X.laevis S-phase chromatin

Project description:Complex cellular processes are driven by the regulated assembly and disassembly of large multi-protein complexes. In eukaryotic DNA replication, whilst we are beginning to understand the molecular mechanism for assembly of the replication machinery (replisome), we still know little about the regulation of its disassembly at replication termination. Over recent years, the first elements of this process emerged: the replicative helicase at the heart of the replisome is polyubiquitylated prior to unloading and that this unloading requires p97 segregase activity. Two different E3 ubiquitin ligases are now known to ubiquitylate the helicase under different conditions: Cul2Lrr1 and TRAIP. To understand how p97 is targeted to the terminated replisome, we immunoprecipitated p97 from chromatin replicating in Xenopus laevis egg extract. We have found a p97 cofactor, Ubxn7, which can interact with active Cul2Lrr1 and with p97, facilitating efficient recognition and processing of terminated helicases ubiquitylated by Cul2.

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