WEHI Special Structural Biology Seminar hosted by Dr Shabih Shakeel

Dr Jacob Lewis

Senior Lecturer of Cell and Molecular Biology, School of Chemistry and Molecular Bioscience – University of Wollongong

Mechanism of replication origin melting nucleated by CMG helicase assembly

L7W Seminar Room (speaker online)

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Including Q&A session

Genome duplication is essential for cell proliferation. Errors in the mechanisms that control DNA replication can cause genomic instability and lead to the development of genetic diseases and cancer. To initiate genome duplication, the MCM helicase is first loaded onto duplex DNA as an inactive double hexamer at origins of replication. Activation occurs upon recruitment of a set of firing factors that assemble two Cdc45-MCM-GINS (CMG) helicases. The mechanistic details by which replicative helicases bind, untwist, and unwind DNA at origins remain elusive, largely due to a lack of high-resolution structures that capture key intermediates during this process. To date, structural studies have focused on imaging artificially isolated replication complexes using simplified DNA substrates to understand mechanisms of DNA replication. We use biochemical and single-particle cryo electron-microscopy (cryo-EM) approaches to visualise origin-dependent eukaryotic CMG assembly using in vitro reconstituted cellular reactions in their entirety, at near atomic resolution. Here, we present the 3.4 Å cryo-EM structure of CMG caught in the act of underwinding origin DNA. Our results provide a structural explanation for previous biochemical experiments on origin-dependent CMG formation in vitro that indicated ATP binding promotes CMG formation, duplex DNA untwisting, and double hexamer separation. We also established how Mcm10 promotes origin unwinding by breaking the double CMGE into two single CMGEs that translocate passed one another and transition from embracing duplex to single-stranded DNA. Visualising DNA melting and origin unwinding elucidates key steps in replication initiation.

Dr Jacob Lewis (PhD, University of Wollongong, 2017) was an EMBO and Marie Curie Fellow (2020-2024, Francis Crick Institute) and currently an ARC DECRA fellow and NHMRC Investigator (University of Wollongong). Dr. Lewis leads a research group at the Molecular Horizons Research Institute, where his team employs advanced cryo-electron microscopy, biochemical techniques, and single-molecule imaging to study molecular machines in action.

His research focuses on understanding how chromosomes are duplicated to ensure accurate genetic information transfer during cell division. This work is crucial for uncovering how DNA maintains its integrity and identifying disruptions that can lead to various human diseases.

All Welcome!