Type of Document Dissertation Author Yang, Yanling Author's Email Address firstname.lastname@example.org URN etd-08042011-230059 Title Thermodynamics of DNA Binding and Break Repair by the Pol I DNA Polymerases from Escherichia coli and Thermus aquaticus Degree Doctor of Philosophy (Ph.D.) Department Biochemistry (Biological Sciences) Advisory Committee
Advisor Name Title LiCata, Vince J. Committee Chair Battista, John R. Committee Member Grove, Anne Committee Member Siebenaller, Joseph F. Committee Member Sehgal, Inder Dean's Representative Keywords
- DNA Binding
- Protein-DNA Interaction
- Gapped DNA
- DNA Double-strand Break Repair
- Mismatched DNA
- Stimulation on Ligase
- 2:1 Polymerase-DNA complex
Date of Defense 2011-07-19 Availability unrestricted AbstractKlenow and Klentaq are the “large fragments” of the Pol I DNA polymerases from Escherichia coli and Thermus aquaticus. Examination of the DNA binding thermodynamics of both polymerases to replication versus repair substrates shows that Klenow binds primed-template DNA with up to 50X higher affinity than it binds to a nicked DNA, gapped DNAs, DNA with blunt-end or a 3’ overhang, while Klentaq binds all of these DNAs similarly. The presence of 5’ or 3’ phosphates has slightly different effects on DNA binding by both polymerases. In contrast, both polymerases bind mismatched DNA tighter than matched DNA, suggesting that they may share a similar mechanism to identify mismatched DNA, despite the lack of proofreading ability in Klentaq.
The effects of Klenow and Klentaq on ligation of DNA ligase were also studied. Both polymerases stimulate the intermolecular ligation activity of E. coli DNA ligase at concentrations sub-stoichiometric to the DNA concentration. This effect occurs with E. coli DNA ligase, but not for T4 and Taq ligases. Additionally, neither polymerase significantly enhances ligation of a substrate containing a single nick, suggesting that the polymerases bridge the two DNA ends during intermolecular ligation.
The nucleotide incorporation activities of both polymerases on substrates minicing double-strand breaks (DSBs) were also examined. Both proteins are able to “repair” DSBs via alignment-based strand-displacement DNA synthesis. Moreover, their repair abilities have different dependences on 5’ phosphate and DNA ligase when DSBs contain non-cohesive ends. Additionally, both proteins mediated palindrome amplification alone when the short inverted repeats occur near DNA breaks, suggesting that short inverted repeats in prokaryotes may help in DSB repair. 5’ phosphate at the matched break end is required for DSBs repair by both polymerases when one break end contains 3 consecutive mismatches.
Results of the electrophoretic mobility shift assay show that Klenow-DNA complexes are observed as slow or fast moving bands, or both while all Klentaq-DNA complexes are observed as slow moving bands. The protection of both ends of a DNA by Klenow from exonuclease digestion suggests that the slow moving bands may correspond to the 2:1 polymerase-DNA complex.
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