Molecular Horizons Seminar - Dr Jacob Lewis

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 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.