Investigating the physiological DNA replication initiation reaction (360G-Wellcome-204985_Z_16_Z)

In all cells chromosome replication requires key initiator proteins to unwind the DNA at specific sites termed origins. Despite the fundamental importance of DNA replication initiation, crucial aspects of the process remain poorly understood. My vision is to identify all essential features of both the bacterial replication origin and DNA replication initiation proteins in vivo. This comprehensive reverse genetic analysis will guide biochemical investigations into the activities associated with deleterious mutations, thereby revealing the interactions and steps necessary for the physiological initiation reaction. Towards this goal I have created a bespoke tool: an inducible heterologous replication initiation system that allows construction and characterization of mutations within endogenous replication initiation factors. This methodology, combined with assays we developed to analyze DNA replication initiation in vitro, led us to identify a new essential bacterial replication origin element (Richardson et al. Nature 2016). We will build upon this successful approach and go on to develop more sophisticated heterologous replication systems, opening the door to studying all aspects of chromosome replication. Importantly, the bacterial DNA replication machinery is an underexploited drug target. Knowledge of bacterial DNA replication resulting from this work will provide a guide for disrupting this process in pathogenic species.