The eukaryotic replisome assembles around the CMG helicase, which stably associates with DNA replication forks throughout elongation. When replication terminates, CMG is ubiquitylated on its Mcm7 subunit and disassembled by the Cdc48 / p97 ATPase. Until now, the regulation that restricts CMG ubiquitylation to termination was unknown, as was the mechanism of disassembly. By reconstituting these processes with purified budding yeast proteins, we show that ubiquitylation is tightly repressed throughout elongation by the Y-shaped DNA structure of replication forks. Termination removes the repressive DNA structure, whereupon long K48-linked ubiquitin chains are conjugated to CMG-Mcm7, dependent on multiple replisome components that bind to the ubiquitin ligase SCFDia2. This mechanism pushes CMG beyond a '5-ubiquitin threshold' that is inherent to Cdc48, which specifically unfolds ubiquitylated Mcm7 and thereby disassembles CMG. These findings explain the exquisite regulation of CMG disassembly and provide a general model for the disassembly of ubiquitylated protein complexes by Cdc48.

F1-ATPase is a rotary molecular motor driven by ATP hydrolysis that rotates the ␥-subunit against the ␣3␤3 ring. The crystal structures of F1, which provide the structural basis for the catalysis mechanism, have shown essentially 1 stable conformational state. In contrast, single-molecule studies have revealed that F1 has 2 stable conformational states: ATP-binding dwell state and catalytic dwell state. Although structural and single-molecule studies are crucial for the understanding of the molecular mechanism of F1, it remains unclear as to which catalytic state the crystal structure represents. To address this issue, we introduced cysteine residues at ␤E391 and ␥R84 of F1 from thermophilic Bacillus PS3. In the crystal structures of the mitochondrial F1, the corresponding residues in the ADPbound ␤ (␤DP) and ␥ were in direct contact. The ␤E190D mutation was additionally introduced into the ␤ to slow ATP hydrolysis. By incorporating a single copy of the mutant ␤-subunit, the chimera F1, ␣3␤2␤(E190D/E391C)␥(R84C), was prepared. In single-molecule rotation assay, chimera F1 showed a catalytic dwell pause in every turn because of the slowed ATP hydrolysis of ␤(E190D/E391C). When the mutant ␤ and ␥ were cross-linked through a disulfide bond between ␤E391C and ␥R84C, F1 paused the rotation at the catalytic dwell angle of ␤(E190D/E391C), indicating that the crystal structure represents the catalytic dwell state and that ␤DP is the catalytically active form. The former point was again confirmed in experiments where F1 rotation was inhibited by adenosine-5-(␤,␥-imino)-triphosphate and/or azide, the most commonly used inhibitors for the crystallization of F1. ATP synthase ͉ cross-link

The eukaryotic replisome is disassembled in each cell cycle, dependent upon ubiquitylation of the CMG helicase. Studies of Saccharomyces cerevisiae, Caenorhabditis elegans and Xenopus laevis have revealed surprising evolutionary diversity in the ubiquitin ligases that control CMG ubiquitylation, but regulated disassembly of the mammalian replisome has yet to be explored. Here, we describe a model system for studying the ubiquitylation and chromatin extraction of the mammalian CMG replisome, based on mouse embryonic stem cells. We show that the ubiquitin ligase CUL2LRR1 is required for ubiquitylation of the CMG‐MCM7 subunit during S‐phase, leading to disassembly by the p97 ATPase. Moreover, a second pathway of CMG disassembly is activated during mitosis, dependent upon the TRAIP ubiquitin ligase that is mutated in primordial dwarfism and mis‐regulated in various cancers. These findings indicate that replisome disassembly in diverse metazoa is regulated by a conserved pair of ubiquitin ligases, distinct from those present in other eukaryotes.