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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. SYNOPSIS: This study reports a model system based on mouse ESC to study the mammalian replisome. It shows that the ubiquitin ligases CUL2LRR1 and TRAIP control p97‐dependent replisome disassembly during DNA replication termination and mitosis, respectively. Mouse embryonic stem cells provide a rich source of the mammalian CMG helicase and associated replisome.CUL2LRR1 is required for p97‐dependent CMG helicase disassembly during DNA replication termination in mouse ES cells.The TRAIP ubiquitin ligase is required for a mitotic pathway of CMG helicase disassembly in mouse ES cells. [ABSTRACT FROM AUTHOR]