The molecular basis of replicative helicase assembly and activation

To duplicate their enormous genomes in a timely manner, eukaryotes initiate DNA replication from multiple sites called origins. To do so, the DNA replication machinery (replisome) is assembled de novo at every origin. This critical process, termed origin firing, involves the highly regulated assembly and activation of the replicative CDC45-MCM-GINS (CMG) helicase that unwinds double-stranded DNA to generate the single-stranded templates needed for DNA synthesis. CMG assembly and activation requires the activity of the kinases DDK and CDK and multiple ‘firing factor’ proteins. Origin firing has been reconstituted with purified yeast proteins. However, due to its complexity, our understanding of the molecular mechanisms that underpin origin firing are incomplete, and it has not been reconstituted with human proteins. The overall objective of this grant proposal is to discover the molecular mechanisms that underpin eukaryotic CMG helicase assembly and activation. To achieve this, we will use our established strategy that combines complex in vitro biochemical reconstitution with purified yeast and human proteins, functional replisome assembly and DNA replication assays, and cryo-EM analysis of DNA-bound replisomes. Our ambitious research programme will transform our understanding of a fundamental aspect of genome maintenance that is critical for human health and disease.