The mechanism of eukaryotic CMG helicase activation Max E Douglas Ferdos Abid Ali Alessandro Costa John FX Diffley 10779/crick.12639233.v1 https://crick.figshare.com/articles/journal_contribution/The_mechanism_of_eukaryotic_CMG_helicase_activation/12639233 The initiation of eukaryotic DNA replication occurs in two discrete stages: first, the minichromosome maintenance (MCM) complex assembles as a head-to-head double hexamer that encircles duplex replication origin DNA during G1 phase; then, 'firing factors' convert each double hexamer into two active Cdc45-MCM-GINS helicases (CMG) during S phase. This second stage requires separation of the two origin DNA strands and remodelling of the double hexamer so that each MCM hexamer encircles a single DNA strand. Here we show that the MCM complex, which hydrolyses ATP during double-hexamer formation, remains stably bound to ADP in the double hexamer. Firing factors trigger ADP release, and subsequent ATP binding promotes stable CMG assembly. CMG assembly is accompanied by initial DNA untwisting and separation of the double hexamer into two discrete but inactive CMG helicases. Mcm10, together with ATP hydrolysis, then triggers further DNA untwisting and helicase activation. After activation, the two CMG helicases translocate in an 'N terminus-first' direction, and in doing so pass each other within the origin; this requires that each helicase is bound entirely to single-stranded DNA. Our experiments elucidate the mechanism of eukaryotic replicative helicase activation, which we propose provides a fail-safe mechanism for bidirectional replisome establishment. 2020-07-15 11:05:00 Adenosine Diphosphate Adenosine Triphosphate Cell Cycle Proteins DNA Helicases DNA Replication DNA, Single-Stranded DNA-Binding Proteins Enzyme Activation Enzyme Stability Minichromosome Maintenance Proteins Nucleic Acid Conformation Replication Origin Saccharomyces cerevisiae Saccharomyces cerevisiae Proteins Diffley FC001066 Costa FC001065 CB-ack EM-ack General Science & Technology