The mechanism of eukaryotic CMG helicase activation
By M. Douglas, F. A. Ali, Alessandro Costa, J. Diffley. 2018
234 citations recorded by the source corpus.
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.
Subfields: Genetics, Molecular Biology
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In Genetics
In Molecular Biology
- Justin L. Sparks
- Matthias Mann
- Johannes C. Walter
- C. Ramsoomair
- Lorraine De Jesús-Kim
- S. Bell
- L. Friedman
- Masato T. Kanemaki
Citations in the corpus
- In silico protein interaction screening uncovers DONSON’s role in replication initiation (2023, background): “RPA is required for origin un-winding and replication elongation ( 31 , 32 ), as seen from defective PCNA and Pol a loading and the persistence of CDC45, GINS, and Pol e on chromatin at the 40-min point (Fig.”
- DDK regulates replication initiation by controlling the multiplicity of Cdc45-GINS binding to Mcm2-7 (2020, methodology): “We simultaneously monitored labeled Mcm2-74SNAP549 and unquenching of the DNA-associated fluorophore in the presence of the proteins required for CMG formation and two additional proteins (Mcm10 and RPA) required for extensive DNA unwinding (Douglas et al., 2018).”
- The CMG helicase bypasses DNA-protein cross-links to facilitate their repair (2018, background): “Given that the non-catalytic N-terminal tier of CMG resides at the leading edge of the fork (Douglas et al., 2018; Georgescu et al., 2017), the N terminus might re-close around ssDNA while the C-terminal ATPase domain is still engaged in bypassing the DPC.”
- DNSN-1 recruits GINS for CMG helicase assembly during DNA replication initiation in C. elegans (2023, unknown): “Recruitment of Cdc45 and GINS triggers the assembly of two CMG helicase complexes (Figure S1, step 5), with their Mcm2-7 rings still encircling double-stranded DNA (22–24).”