Mechanism of replication origin melting nucleated by CMG helicase assembly

By Jacob S. Lewis, Marta H. Gross, Joana Sousa, Sarah S. Henrikus, Julia F. Greiwe, A. Nans, J. Diffley, Alessandro Costa. 2022

83 citations recorded by the source corpus.

The activation of eukaryotic origins of replication occurs in temporally separated steps to ensure that chromosomes are copied only once per cell cycle. First, the MCM helicase is loaded onto duplex DNA as an inactive double hexamer. Activation occurs after the recruitment of a set of firing factors that assemble two Cdc45–MCM–GINS (CMG) holo-helicases. CMG formation leads to the underwinding of DNA on the path to the establishment of the replication fork, but whether DNA becomes melted at this stage is unknown1. Here we use cryo-electron microscopy to image ATP-dependent CMG assembly on a chromatinized origin, reconstituted in vitro with purified yeast proteins. We find that CMG formation disrupts the double hexamer interface and thereby exposes duplex DNA in between the two CMGs. The two helicases remain tethered, which gives rise to a splayed dimer, with implications for origin activation and replisome integrity. Inside each MCM ring, the double helix becomes untwisted and base pairing is broken. This comes as the result of ATP-triggered conformational changes in MCM that involve DNA stretching and protein-mediated stabilization of three orphan bases. Mcm2 pore-loop residues that engage DNA in our structure are dispensable for double hexamer loading and CMG formation, but are essential to untwist the DNA and promote replication. Our results explain how ATP binding nucleates origin DNA melting by the CMG and maintains replisome stability at initiation. Cryo-electron microscopy structures of the sequential assembly of the CMG replicative helicase on a chromatinized origin of replication provide insights into the mechanism through which DNA melting is initiated by ATP binding.

Subfields: Molecular Biology

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In Molecular Biology

1. Johannes C. Walter
2. Masato T. Kanemaki
3. Sachiko Muramatsu

Citations in the corpus

• In silico protein interaction screening uncovers DONSON’s role in replication initiation (2023, background): “We speculate that disengagement andclockwiserotation ofthetwo MCMs, as seen during CMG assembly in yeast ( 45 ), might enable DONSON dimer binding to both MCM3 molecules simultaneously (fig.”
• DNSN-1 recruits GINS for CMG helicase assembly during DNA replication initiation in C. elegans (2023, unknown): “Subsequently, the Mcm10 protein is required for a poorly understood step in which the Mcm2-7 ring is opened transiently to exclude one DNA strand (22, 23), leading to full activation of the helicase (Figure S1, step 6).”