Origin-dependent DNA replication visualised by Cryo-EM

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.

In vitro reconstitution of DNA replication with purified yeast proteins has helped uncover important mechanisms of DNA replication.

How changes in protein structure regulates function during key events in origin activation and replisome progression, such as melting of the DNA duplex upon CMG helicase assembly, or the mode of binding of Pol alpha during primer synthesis remain unknown.

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.

To address this issue, I will integrate single-particle cryo electron-microscopy (cryo-EM) with sophisticated biochemical approaches to image origin-dependent DNA replication reactions in vitro on native DNA substrates.

To do so, I will establish short origin-containing DNA substrates that permit loading of a single bidirectional replication fork, allowing large numbers of protein bound origins to be visualised within a single field of view. Initially, I will investigate how the structure of duplex DNA changes upon CMG formation during origin activation.

Next, I will examine the molecular mechanisms of primer synthesis after origin activation using both cryo-EM and in vitro DNA replication reactions. Finally, I will capture and image synthesising intact replisomes at near atomic resolution.

By visualising entire DNA replication reactions instead of isolated replication complexes at high resolution, we will gain a deeper understanding of the molecular mechanisms that permit the eukaryotic replisome to function during genome duplication.