Doctor of Philosophy (PhD)
Deinococcus radiodurans has a significantly more robust DNA repair response than Escherichia coli, which helps it survive high amounts of oxidative stress. D. radiodurans DNA Polymerase I (DrPolI) and RecA protein (DrRecA) play essential parts in this DNA repair capability. Structural stability studies of the DrPolI large fragment (Klendra) and examination of the DNA binding characteristics of Klendra and RecA are presented herein. In the absence of cofactors DrRecA binds to dsDNA more than 20 fold tighter than EcRecA, and binds ssDNA up to 9 fold tighter. Binding to dsDNA in the absence of cofactor presumably monitors DNA end binding. ATPãS abolishes the species-specific affinity difference. Both proteins possess dependence of binding affinity on the length of ssDNA, but not the length of dsDNA, which results in tighter binding to ssDNA at increasing lengths. These studies indicate an enhanced binding to DNA ends during periods of metabolic stress, which could be important for repair responses after incursion of DNA damage. Klendra polymerase displays an overall increased stability and DNA binding capability as solution pH is brought to 5.75. Klendra has a Tm at pH 5.75 of 46oC, and a ÄGunfolding at pH 7.9 of 8-10 Kcal/mol. Klendra is stable over a wide range of pH from 3-11, potentially reflecting a necessity of DNA polymerase I functionality at lower pH during DNA repair. This idea is further supported by the enhancement in binding affinity for Klendra at pH 5.75, where it binds both ptDNA and dsDNA with high affinity. Klendra displays proton uptakes upon binding to dsDNA and ptDNA of 0.9 H+ ions and 1.4 H+ ions, respectively, and has higher affinity for dsDNA over ptDNA over a wide range of pH and KCl conditions. A non-linear ptDNA KCl linkage results in little selectivity of dsDNA over ptDNA at KCl concentrations above 50mM. Klendra lacks the ability to bind ssDNA, potentially due to absence of proofreading activity.
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Warfel, Jaycob Dalton, "Thermodynamics of DNA Binding by DNA Polymerase I and RecA Recombinase from Deinococcus radiodurans" (2014). LSU Doctoral Dissertations. 2382.