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