The accurate transfer of genetic information from parental to daughter cells requires that the genome be faithfully copied only once per cell cycle. Failures at any point in this process can lead to cellular abnormalities, genetic disease, and cancer. DNA replication is catalysed by the replisome, a large multi-protein complex that coordinates DNA unwinding and synthesis. A hierarchy of strong and weak functional pair-wise interactions controls and maintains the replisome allowing it to transition through multiple conformational states. These transitions are intimately linked to a small number of essentially irreversible chemical steps (ATP-hydrolysis, RNA-primer and DNA synthesis), which determine reaction directionality. In vitro reconstitution of DNA replication with purified yeast proteins has helped uncover important mechanisms of DNA replication. To date, structural studies have focused on imaging artificially isolated replication complexes using simplified DNA substrates to understand DNA unwinding and replisome architecture. To truly understand the mechanisms that control DNA replication, future structural studies must not only visualise isolated complexes, but also reconstituted reactions. This project aims to visualise origin-dependent eukaryotic DNA replication using in vitro reconstituted cellular reactions in their entirety, at near atomic resolution. These data will gain a deeper understanding of the molecular mechanisms that permit the eukaryotic replisome to function during genome duplication.
This project has shed some valuable light on the previously unknown mechanism of origin DNA unwinding and spatial orientation of key replisome components.
