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dc.contributor.advisorBrian A. Kelch, PhD
dc.contributor.authorDuffy, Caroline M.
dc.date2022-08-11T08:08:46.000
dc.date.accessioned2022-08-23T16:07:17Z
dc.date.available2022-08-23T16:07:17Z
dc.date.issued2016-07-15
dc.date.submitted2016-11-28
dc.identifier.doi10.13028/M22P45
dc.identifier.urihttp://hdl.handle.net/20.500.14038/32217
dc.description.abstractChromosomal replication is an essential process in all life. This dissertation highlights regulatory roles for two critical protein complexes at the heart of the replication fork: 1) the sliding clamp, the major polymerase processivity factor, and 2) the sliding clamp loader, a spiral-shaped AAA+ ATPase, which loads the clamp onto DNA. The clamp is a promiscuous binding protein that interacts with at least 100 binding partners to orchestrate many processes on DNA, but spatiotemporal regulation of these binding interactions is unknown. Remarkably, a recent disease-causing mutant of the sliding clamp showed specific defects in DNA repair pathways. We aimed to use this mutant as a tool to understand the binding specificity of clamp interactions, and investigate the disease further. We solved three structures of the mutant, and biochemically showed perturbation of partnerbinding for some, but not all, ligands. Using a fission yeast model, we showed that mutant cells are sensitive to select DNA damaging agents. These data revealed significant flexibility within the binding site, which likely regulates partner binding. Before the clamp can act on DNA, the sliding clamp loader places the clamp onto DNA at primer/template (p/t) junctions. The clamp loader reaction couples p/t binding and subsequent ATP hydrolysis to clamp closure. Here we show that composition (RNA vs. DNA) of the primer strand affects clamp loader binding, and that the order of ATP hydrolysis around the spiral is likely sequential. These studies highlight additional details into the clamp loader mechanism, which further elucidate general mechanisms of AAA+ machinery.
dc.language.isoen_US
dc.rightsCopyright is held by the author, with all rights reserved.
dc.subjectDissertations, UMMS
dc.subjectDNA Replication
dc.subjectDNA-Binding Proteins
dc.subjectDNA Primers
dc.subjectDNA Replication
dc.subjectDNA-Binding Proteins
dc.subjectDNA Primers
dc.subjectAmino Acids, Peptides, and Proteins
dc.subjectBiochemistry
dc.subjectMolecular Biology
dc.subjectStructural Biology
dc.titleStructural Mechanisms of the Sliding Clamp and Sliding Clamp Loader: Insights into Disease and Function: A Dissertation
dc.typeDoctoral Dissertation
dc.identifier.legacyfulltexthttps://escholarship.umassmed.edu/cgi/viewcontent.cgi?article=1858&context=gsbs_diss&unstamped=1
dc.identifier.legacycoverpagehttps://escholarship.umassmed.edu/gsbs_diss/844
dc.legacy.embargo2017-07-22T00:00:00-07:00
dc.identifier.contextkey9416791
refterms.dateFOA2022-08-29T14:22:51Z
html.description.abstract<p>Chromosomal replication is an essential process in all life. This dissertation highlights regulatory roles for two critical protein complexes at the heart of the replication fork: 1) the sliding clamp, the major polymerase processivity factor, and 2) the sliding clamp loader, a spiral-shaped AAA+ ATPase, which loads the clamp onto DNA.</p> <p>The clamp is a promiscuous binding protein that interacts with at least 100 binding partners to orchestrate many processes on DNA, but spatiotemporal regulation of these binding interactions is unknown. Remarkably, a recent disease-causing mutant of the sliding clamp showed specific defects in DNA repair pathways. We aimed to use this mutant as a tool to understand the binding specificity of clamp interactions, and investigate the disease further. We solved three structures of the mutant, and biochemically showed perturbation of partnerbinding for some, but not all, ligands. Using a fission yeast model, we showed that mutant cells are sensitive to select DNA damaging agents. These data revealed significant flexibility within the binding site, which likely regulates partner binding.</p> <p>Before the clamp can act on DNA, the sliding clamp loader places the clamp onto DNA at primer/template (p/t) junctions. The clamp loader reaction couples p/t binding and subsequent ATP hydrolysis to clamp closure. Here we show that composition (RNA vs. DNA) of the primer strand affects clamp loader binding, and that the order of ATP hydrolysis around the spiral is likely sequential. These studies highlight additional details into the clamp loader mechanism, which further elucidate general mechanisms of AAA+ machinery.</p>
dc.identifier.submissionpathgsbs_diss/844
dc.contributor.departmentBiochemistry and Molecular Pharmacology
dc.description.thesisprogramBiochemistry and Molecular Pharmacology


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