Fragment-based interrogation of a bacterial replisome

Chromosomes are replicated by the replisome, a molecular machine comprised of a set of interacting proteins with interdependent functions. In previous work, a fragment library was screened in vitro against a bacterial replisome, reconstituted from thirteen different subunits. In this thesis, the results of this phenotypic assay were validated, analysed and extended with functional and biophysical experiments to explore different mechanisms of inhibition. �e work shows that phenotypic fragment screening can be used as a starting point for target identification. Assays for the individual protein complexes in the replisome revealed that most fragment inhibitors acted through specific proteins. For one fragment inhibitor, the results were consistent with inhibition of primase, an enzyme that synthesises short primers to initiate elongation. �e mode of action of this fragment and analogues were studied in detail. �e bacterial primase consists of three domains: a helicase-binding, a zinc-binding, and a polymerase domain. �e fragment was not observed to bind to the polymerase domain in X-ray crystallography and nuclear magnetic resonance experiments. Molecular dynamics simulations showed a clear preference for binding to a conserved protein-protein interaction site on the helicase-binding domain, rather than to the zincbinding domain. �is prediction was validated by protein-observed nuclear magnetic resonance experiments and structure-activity and -affinity relationships. Some of the replisome fragment inhibitors acted only on the full machine and do not show inhibition of the available assays for individual components. Methods were developed to generate and test photoreactive fragment hit analogues and to screen covalent cysteine-targeting fragments. �is work did not identify any new site of interaction.