Thesis (Ph.D.)--Washington State University, December 2005. / Includes bibliographical references.

Thesis (Ph.D.) - University of St Andrews, May 2008.

Thesis (Ph.D.) - University of St Andrews, April 2009.

Thesis (Ph. D.)--Ohio State University, 2007. / Full text release at OhioLINK's ETD Center delayed at author's request

No description available.

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Apurinic endonuclease activity in human fibroblasts had been previously resolved into aflow-through and a high-salt eluate species by phosphocellulose chromatography (Kuhnlein, U. et al., Nucl. Acid. Res. 5: 951-960, 1978). Enzyme activity in the flow-through species amounted to 20-30% that of the high-salt eluate species. The flow—through enzyme species was not found in-eel I lines of xeroderma pigmentosum complementation group D. In this thesis, apurinic endonuclease activity was analysed in Hela ceflls. Specific enzyme activity in crude extracts "of Hela cells was in the range of 400-800 units/mg protein, similar to that of , human fibroblasts which was between 380-680 units/mg protein. Three species of endonuclease activity for apurinic DNA were resolved by phosphocellulose chromatography. They were designated as Peak I, Peak If, and Peak III. Peak I did not adsorb to the phosphoceIIuIose column at' 10 mM KP04 (pH 7.4) (flow-through activity), Peak II eluted from the column at about 210 mM KP0₄ (pH 7.4) and Peak I I I at 260 mM KPO₄ (pH 7.4). Based on their affinity to phosphoceIIuIose, we presumed Peak I and Peak III corresponded to the flow-through and high-salt eluate species in human fibroblasts respectively. Under our experimental conditions, the flow-through enzyme activity in both Hela cells and normaS human fibroblasts was only 2-4% of the activity of high-salt eluate species. We suspect that tissue culture conditions may affect the cellular level of the flow-through species of apurinic endonuclease. Peaks l-lII were optimally active at pH 7.5-8.0 and 5-10 mM MgCI, They were 'inhibited by increasing concentrations of KCI and NaCI except Peak III which was slightly stimulated by 20-40 mM KCI. The three species were distinguished by their thermosensitivities in a 50 mM KPO. buffer. Peak I was stable at 45°C. Peak III was heat-labile, having a half-life of 2-3 min at 45°C. Peak II seemed to contain two components, one with a ha If-life of 2-3 min at 45°C, and the other with a half-life of 25 min. In human fibroblasts, both the flow-through and high-salt eluate species of apu-rinic endonuclease were reported to be stimulated to 2.5-fold by 10 mM KCI. They had a ha If-life of 6 min at 45°C in a 230 mM KP0₄ (pH 7.4) buffer. Thus, Peaks I-lI I and enzyme species from human fibroblasts had a similar pH optimum, and Mg²⁺ requirement, but they differed in their thermosensitivities and inhibition by higher salt concentration. We do not know as yet whether these differences reflect the neoplastic nature of Hela cells or the different tissue origins of Hela cells and human fibroblasts. When either Peak I or Peak I I I was rechromatographed on the phosphoceIIulose column, activity was recovered in both the flow-through and high-salt eluate fractions. The result suggested an interconversion phenomenon between the flow-through and high-salt eluate species of apurinic endonuclease, This was further supported by molecular weight determinations of the apurinic endonucI eases In Peaks l-lII. Apurinic endonuclease activity in Peak III and Peak II had a molecular weight of 35,000-40,000 and 22,000-25,000 respectively. Peak I had two components with molecular weights similar to those of Peak II and Peak III. An understanding of the conversion between the different apurinic endonuclease species may help in elucidating the molecular defects of xeroderma pigmentosum complementation group D. Apurinic endonuclease activity in Peaks l-lII was found to be associated with a high molecular weight complex. The complex could be dissociated by high salt treatment. The possible biological significance of the high molecular weight complex Is discussed. We also found that apurinic endonuclease could adsorb to the Sephadex gel. The adsorption would lead to an aberrant estimation of molecular weight of the protein. The problem was solved with an elution buffer of high ionic strength. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

No description available.

DNA double strand breaks represent the single most dangerous type of damage that can afflict the genome. Given the severity of such a lesion, higher eukaryotes possess two distinct pathways to repair such damages. The work presented here focuses on the role of different protein complexes formed during within Non-Homologous End Joining (NHEJ). Specifically, how the C-terminal tails of XRCC4 and XLF regulate higher-order complex formation of end bridging filaments prior to terminal ligation and release of the intact DNA following repair. The crystal structure of full length XRCC4 was solved to 3.43Å and confirmed that the C-terminal tails or XRCC4 mediate tetramerization but are not required for end bridging of DNA ends. A cluster of residues that stabilized the XRCC4 multi helix bundle were mutated and determined to result in an XRCC4Mutational analysis and SEC-MALS further revealed that this 4-helix bundle stabilizes tetramers, interestingly tetramerization was found to not be required for bridging of DNA ends. Additional work aimed at determining the mechanism by which XLF binds DNA and how complex filaments are formed was carried out using a combination of structural and biochemical techniques. Mutational analysis of the yeast XLF homologue, Nej1, revealed that the tails of these proteins bind their DNA substrates through an extended interface that may involve wrapping DNA to further stabilize interaction. Also, it was determined that phosphorylation of key residues within this extended DNA binding domain results in a decreased affinity for DNA and may play a role in DNA repair pathway choice in Saccharomyces cerevisiae. Transmission electron microscopy showed that when bound to DNA, XLF is capable of forming DNA dependent filaments that are capable of bridging DNA ends in a linear manner. Addition of XRCC4 resulted in extensive remodelling of these filaments. Crystals of the XRCC4/XLF/DNA were optimized to diffract to a resolution of greater than 5Å, however further work will be required to determine the structure of this key NHEJ complex. Finally, attempts to determine the optimal combination of DNA substrates and NHEJ factors to crystallize the terminal repairosome were carried out and initial hit conditions have been identified. / Thesis / Doctor of Philosophy (PhD)