In Vitro Reconstitution Defines the Minimal Requirements for Cdc48-Dependent Disassembly of the CMG Helicase in Budding Yeast

• Published in: Cell reports (Cambridge) 2019-09, Vol.28 (11), p.2777-2783.e4
• Main Authors: Mukherjee, Progya P., Labib, Karim P.M.
• Format: Article
• Language: English
• Subjects: ATPase; Cdc48; CMG helicase; DNA Helicases - genetics; DNA Helicases - metabolism; DNA replication; DNA Replication - genetics; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; F-Box Proteins - metabolism; In Vitro Techniques; Mcm7; Minichromosome Maintenance Complex Component 7 - genetics; Minichromosome Maintenance Complex Component 7 - metabolism; Nuclear Proteins - genetics; Nuclear Proteins - metabolism; Nucleocytoplasmic Transport Proteins - genetics; Nucleocytoplasmic Transport Proteins - metabolism; p97; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Saccharomyces cerevisiae - enzymology; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae Proteins - genetics; Saccharomyces cerevisiae Proteins - metabolism; SCFDia2; Ubiquitination - genetics; ubiquitylation; Ufd1-Npl4; Valosin Containing Protein - genetics; Valosin Containing Protein - metabolism; Vesicular Transport Proteins - genetics; Vesicular Transport Proteins - metabolism
• ISSN: 2211-1247; 2211-1247
• DOI: 10.1016/j.celrep.2019.08.026

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 SCFDia2. 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 replisome disassembly that are observed in vivo. Our findings define the minimal requirements for disassembly of the eukaryotic replisome and provide a model system for studying the disassembly of protein complexes by Cdc48-Ufd1-Npl4. [Display omitted] •In vitro ubiquitylation of budding yeast replisome by SCFDia2•SCFDia2 ubiquitylates the CMG helicase without priming by HECT or RBR ligases•In vitro reconstitution of the disassembly of ubiquitylated CMG•Budding yeast Cdc48-Ufd1-Npl4 are sufficient to disassemble ubiquitylated CMG To study the mechanism of CMG helicase disassembly during DNA replication termination, Mukherjee and Labib purify a complex of the yeast replisome with the E3 ligase SCFDia2. After in vitro ubiquitylation of the Mcm7 subunit of CMG, recombinant Cdc48 with its adaptors Ufd1-Npl4 are sufficient to drive efficient CMG disassembly.