Journal Article

The mechanism of eukaryotic CMG helicase activation

Costa, Alessandro, Douglas, Max E., Diffley, John F. X., Ali, Ferdos Abid

2018

Overview

In vitro experiments, using purified proteins and an assay that detects DNA unwinding, reveal the mechanism of activation of eukaryotic DNA replication. Unravelling DNA replication DNA replication in eukaryotes begins with the loading of a double hexamer of minichromosome maintenance (MCM) proteins onto the origin. Replication is then activated by separating the double hexamer into single-hexamer MCM rings that, together with Cdc45 and GINS, make up the CMG helicase, which is required for DNA unwinding. John Diffley and colleagues describe the role of ATP hydrolysis in regulating double-hexamer assembly and then CMG formation. Notably, there is an inactive CMG state that precedes the helicase-active CMG form that can translocate along the unwound DNA strand. The active CMG moves unidirectionally so that the two helicases pass by each other to establish bidirectional replication. The initiation of eukaryotic DNA replication occurs in two discrete stages 1 : 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 2 , 3 , 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.

• Publisher: Nature Publishing Group UK,Nature Publishing Group
• Subject: 101/28 / 631/337/151/2355 / 631/45/147 / 631/45/173 / 82 / 82/1 / 82/16 / 82/29 / 82/80 / 82/83 / Activation / Adenosine diphosphate / Adenosine Diphosphate - chemistry / Adenosine Diphosphate - metabolism / Adenosine Triphosphate - chemistry / Adenosine Triphosphate - metabolism / Assembly / Cdc45 protein / Cell Cycle Proteins - metabolism / Deoxyribonucleic acid / DNA / DNA biosynthesis / DNA helicase / DNA Helicases - chemistry / DNA Helicases - metabolism / DNA Replication / DNA, Single-Stranded - biosynthesis / DNA, Single-Stranded - chemistry / DNA, Single-Stranded - metabolism / DNA-Binding Proteins - metabolism / Enzyme Activation / Enzyme Stability / G1 phase / Humanities and Social Sciences / letter / Microscopy / Minichromosome Maintenance Proteins - metabolism / multidisciplinary / Nucleic Acid Conformation / Observations / Replication / Replication initiation / Replication Origin / Replication origins / S phase / Saccharomyces cerevisiae - enzymology / Saccharomyces cerevisiae Proteins - chemistry / Saccharomyces cerevisiae Proteins - metabolism / Science / Separation / Single-stranded DNA