biostudies-literatureMukherjee PPMedical Research CouncilWellcome Trust2777-2783.e4https://www.ebi.ac.uk/biostudies/studies/S-EPMC6899518biostudies-literatureUnknown28(11)Disassembly of the replisome is the final step of chromosome duplication in eukaryotes. In budding yeast and metazoa, cullin ubiquitin ligases are required to ubiquitylate the Cdc45-MCM-GINS (CMG) helicase that lies at the heart of the replisome, leading to a disassembly reaction that is dependent upon the ATPase known as Cdc48 or p97. Here, we describe the reconstitution of replisome disassembly, using a purified complex of the budding yeast replisome in association with the cullin ligase SCF^Dia2. Upon addition of E1 and E2 enzymes, together with ubiquitin and ATP, the CMG helicase is ubiquitylated on its Mcm7 subunit. Subsequent addition of Cdc48, together with its cofactors Ufd1-Npl4, drives efficient disassembly of ubiquitylated CMG, thereby recapitulating the steps of repliCell reportsIn Vitro Reconstitution Defines the Minimal Requirements for Cdc48-Dependent Disassembly of the CMG Helicase in Budding Yeast.PMC6899518MC_UU_00018/4MC_UU_12016/13102943/Z/13/ZMukherjee PPLabib KPMfalseIn Vitro Reconstitution Defines the Minimal Requirements for Cdc48-Dependent Disassembly of the CMG Helicase in Budding Yeast.Disassembly of the replisome is the final step of chromosome duplication in eukaryotes. In budding yeast and metazoa, cullin ubiquitin ligases are required to ubiquitylate the Cdc45-MCM-GINS (CMG) helicase that lies at the heart of the replisome, leading to a disassembly reaction that is dependent upon the ATPase known as Cdc48 or p97. Here, we describe the reconstitution of replisome disassembly, using a purified complex of the budding yeast replisome in association with the cullin ligase SCF^Dia2. Upon addition of E1 and E2 enzymes, together with ubiquitin and ATP, the CMG helicase is ubiquitylated on its Mcm7 subunit. Subsequent addition of Cdc48, together with its cofactors Ufd1-Npl4, drives efficient disassembly of ubiquitylated CMG, thereby recapitulating the steps of repli2019-01-01T00:00:00Z2019 Sep2025-04-04T14:59:49.227Z2025-04-04T14:59:49.227ZS-EPMC68995183150974110.1016/j.celrep.2019.08.026