Hogle, James M.Kath, James Evon2016-04-212016-032015-11-132016Kath, James Evon. 2016. DNA Polymerase Exchange and Lesion Bypass in Escherichia Coli. Doctoral dissertation, Harvard University, Graduate School of Arts & Sciences.http://nrs.harvard.edu/urn-3:HUL.InstRepos:26718716Translesion synthesis (TLS) alleviates replication stalling at DNA lesions. Bypass of lesions by specialized translesion DNA polymerases involves exchange with high-fidelity replicative polymerases. As a consequence of their lesion bypass activity, TLS polymerases are mutagenic, requiring careful regulation of polymerase selection. In this dissertation, I describe a single-molecule reconstitution of polymerase exchange and lesion bypass. Using Escherichia coli polymerases as a model system, I have determined that the dimeric processivity clamp can simultaneously bind a replicative polymerase and a translesion polymerase, facilitating rapid exchange during synthesis and lesion bypass. Overlapping sets of polymerase-clamp interactions additionally allow the TLS polymerase Polymerase IV to displace the replicative polymerase Polymerase III. I finally describe the observation of single Polymerase IV molecules in living cells and initial efforts to determine their localization and dynamics during TLS.application/pdfenBiophysics, GeneralChemistry, BiochemistryBiology, MicrobiologyThesis or Dissertation2017-03-01