Brian KelchLandeck, Jacob T2024-04-302024-04-302024-04-0110.13028/9ba1-hd19https://hdl.handle.net/20.500.14038/53325The sliding clamp is an integral protein in DNA replication and repair, where it increases the speed of DNA synthesis and serves as a scaffold for repair proteins. Since the sliding clamp is a closed ring, clamp loaders must open the ring and then close it around DNA. The clamp loader and sliding clamp are the only two components of the replisome that are conserved across all three domains of life. However, differences in their structures and biochemical activities suggest that the mechanisms of opening and loading differ between prokaryotes and eukaryotes. Structures of the eukaryotic clamp loader clearly illustrate its clamp loading mechanism, but there were no comparable structures for bacterial clamp loaders. To understand how mechanisms of bacterial clamp loaders compare to their eukaryotic counterparts, I determined a series of structures of the E. coli clamp loader at distinct stages in clamp loading. To understand how ATP binding enables the clamp loader to bind to the sliding clamp, I determined a structure of the E. coli clamp loader bound to a non-hydrolyzable ATP analog. I found that the E. coli clamp loader opens its sliding clamp at a single pivot point into a planar conformation, but transitions to a helical conformation upon binding primer-template (p/t)-junctions. This behavior contrasts with eukaryotic clamp loaders, which open their sliding clamp through multiple pivot points into a helical conformation before binding p/t-junctions. My work also revealed that like the eukaryotic clamp loader, the E. coli clamp loader does not need to undergo a conformational change to close the sliding clamp on p/t-junctions. Furthermore, I explored how the E. coli clamp loader is inhibited by the bacteriophage protein gene product 8. This dissertation explores the structural mechanisms used by the bacterial clamp loader and illuminates similarities and differences between clamp loaders across the domains of life.en-USCopyright © 2024 Jacob Landeckhttps://creativecommons.org/licenses/by-nc/4.0/Cryo-EMDNA replicationStructural BiologyClamp LoaderSliding ClampGamma ComplexBeta ClampDoctoral Dissertation0000-0001-5166-1907