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DONSON is one of 13 genes mutated in a form of primordial microcephalic dwarfism known as Meier‐Gorlin syndrome. The other 12 encode components of the CDC45‐MCM‐GINS helicase, around which the eukaryotic replisome forms, or are factors required for helicase assembly during DNA replication initiation. A role for DONSON in CDC45‐MCM‐GINS assembly was unanticipated, since DNA replication initiation can be reconstituted in vitro with purified proteins from budding yeast, which lacks DONSON. Using mouse embryonic stem cells as a model for the mammalian helicase, we show that DONSON binds directly but transiently to CDC45‐MCM‐GINS during S‐phase and is essential for chromosome duplication. Rapid depletion of DONSON leads to the disappearance of the CDC45‐MCM‐GINS helicase from S‐phase cells and our data indicate that DONSON is dispensable for loading of the MCM2‐7 helicase core onto chromatin during G1‐phase, but instead is essential for CDC45‐MCM‐GINS assembly during S‐phase. These data identify DONSON as a missing link in our understanding of mammalian chromosome duplication and provide a molecular explanation for why mutations in human DONSON are associated with Meier‐Gorlin syndrome. Synopsis: DONSON is required for assembly of the CMG helicase in mammalian cells. This explains why hypomorphic mutations in human DONSON cause Meier‐Gorlin Syndrome, which is associated with defects in CMG helicase assembly or function. DONSON binds directly to the CMG helicase but is not a stable component of the replisome.DONSON is essential for DNA replication in mouse embryonic stem cells.DONSON is essential for assembly of the CMG helicase.DONSON is dispensable for loading of the MCM2‐7 helicase core but is required for stable association of GINS and CDC45 with loaded MCM2‐7. [ABSTRACT FROM AUTHOR]

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]