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INTRODUCTION: Eukaryotic chromosomes are duplicated by a molecular machine known as the replisome, the assembly of which is highly regulated to ensure that cells make a single copy of their genome during each cell cycle. In humans, defects in replisome assembly are often associated with early cancer development and can lead to a form of microcephalic primordial dwarfism called Meier-Gorlin syndrome. Eukaryotic replisome assembly is initiated by the assembly and activation of the 11-subunit helicase called CMG (CDC-45–MCM-2-7–GINS), around which the replisome forms. First, two rings of the six adenosine triphosphatases (ATPases) known as MCM-2-7 (comprising the MCM-2 to MCM-7 proteins) are assembled around double-stranded DNA to form double hexamers at replication origins at the end of mitosis. Second, CDC-45 and the four-protein GINS complex are recruited to MCM-2-7 double hexamers during S phase to form a pair of CMG helicases at the heart of two nascent replisomes, in a process that is controlled by several protein kinases and multiple assembly factors. Finally, the two helicases are activated in a poorly understood step, in which the MCM-2-7 rings are opened transiently to exclude one DNA strand. RATIONALE: The mechanism of CMG helicase assembly and activation has been studied most intensively using budding yeast, for which the entire cycle of DNA replication has been reconstituted with purified proteins. However, recent evidence indicates considerable evolutionary diversification in the factors that mediate and control CMG assembly. For example, the Cdc7 kinase is essential for helicase assembly in budding yeast, yet CDC7 is dispensable in untransformed mouse and human cells. Furthermore, the yeast Sld2 protein is an essential helicase assembly factor with homology to vertebrate RECQL4, but studies of Xenopus RECQL4 indicate that it acts after CMG assembly. These findings suggest that animal cells contain additional helicase assembly factors that remain to be identified. RESULTS: Using the embryo of the nematode Caenorhabditis elegans as a model for studying metazoan replisome assembly, we show that a protein called DNSN-1 (ortholog of human DONSON) is essential for the initiation of DNA replication but is dispensable for the subsequent progression of replication forks. Our data indicate that the BRCT-repeat protein MUS-101/TOPBP1 recruits DNSN-1 to the preinitiation complexes that form on MCM-2-7 double hexamers during S phase, thereby triggering assembly of the CMG helicase. We show that DNSN-1 is dispensable for recruitment of CDC-45 to chromatin but is essential for the loading of GINS. Cryo–electron microscopy demonstrates that a dimer of DNSN-1 binds simultaneously to multiple sites on GINS and the MCM-3 helicase subunit, indicating that DNSN-1 positions GINS to promote CMG assembly during DNA replication initiation. Consistent with this view, we show that deletion of one of the binding sites on DNSN-1 for GINS leads to initiation defects, whereas point mutations in DNSN-1 that disrupt one of the interfaces with MCM-3 are lethal and block recruitment of DNSN-1 to preinitiation complexes during S phase. In budding yeast, the Mcm10 protein is required for activation of the nascent CMG helicase complexes. We show that near-complete deletion of the mcm-10 coding sequence by CRISPR-Cas9 is viable in C. elegans, indicating that other factors can also contribute to helicase activation in metazoa. Our data suggest that DNSN-1 might also play a role during this step, because RNAi depletion of DNSN-1 is synthetic lethal with mcm-10D. Orthologs of DNSN-1 are found in animals and plants but are absent from budding yeast and many fungal species. This suggests that the role of DNSN-1/DONSON during DNA replication initiation emerged at an early step of eukaryotic evolution but was subsequently lost during fungal evolution. CONCLUSION: Our findings identify DNSN-1/ DONSON as a missing link in our understanding of DNA replication initiation in animal cells, with an essential role in CMG helicase assembly. The requirement for DNSN-1 during DNA replication initiation indicates substantial differences in the mechanism of replisome assembly between metazoa and budding yeast. Consistent with the essential role of DNSN-1 during CMG helicase assembly in C. elegans, mutations in human DONSON lead to Meier-Gorlin syndrome.
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- 1 Finished
1/03/18 → 1/09/23