| Everyday activities, such as walking outside under the sun or eating barbequed meat, can be detrimental to genomic stability if proper cellular defense mechanisms do not function. The nucleotide excision repair (NER) machinery is the main defense mechanism against bulky DNA damage caused by mainly exogenous mutagens, including ultraviolet light from sun causing two adjacent thymidine residues in DNA to form a dimer and a polycyclic aromatic hydrocarbon compound benzopyrene found in burnt food and cigarette smoke. Unlike many other DNA repair enzymes, each of which acts upon a specific lesion, the NER can recognize and catalyze the removal of a diverse range of lesions in a multi-component and multi-step manner. In bacteria, three protein components, UvrA, UvrB, and UvrC, are involved in damage recognition and removal. UvrA plays a critical role in damage recognition, whereas UvrC participates in damage removal by incising two distinct sites flanking a lesion. Unlike UvrA and UvrC, UvrB interacts with UvrA first, and then DNA and UvrC, therefore it is involved in all phases of the NER repair cascade. Despite intense biochemical studies since its discovery in the sixties, structural characterization of the NER components, either apoprotein or protein-DNA complexes, have been limited to UvrB and the N-terminal domain of UvrC.; The main part of this thesis describes the structure of Bacillus stearothermophilus full-length UvrA solved to 3.2 A resolution using selenium MAD phasing and zinc SAD phasing techniques. The structure provides new insights into the potential mode of UvrA dimer formation as well as roles of zinc finger motifs and ABC ATPase domains in the protein. Additionally, it reveals the positions of the zinc atoms, which allows us to formulate new biochemically testable hypotheses on the functional roles of those sites.; From biochemical studies using E. coli, it is concluded that UvrC is responsible for incision at both sides of a lesion, and 3' incision occurs first followed by 5' incision. In our model system B. st., however, 3' incision is deficient while 5' incision occurs without the necessary 3' incision. The first example of this unusual incision mechanism was published only recently, and B. st. is the second organism that does not seem to possess the efficient 3' incision activity.; It is clear from the UvrA structure and other structures available, it is necessary to capture interacting protein-protein and protein-DNA complexes and structurally characterize those in order to fully comprehend the functional mechanism by which UvrABC repairs a wide range of DNA damage. Attempts on UvrC-CTD apoprotein, a functional fragment responsible for 5' incision, and UvrA-DNA complex crystallization are made. With the UvrC-CTD initial needle crystals are obtained, but they are not diffraction quality crystals. UvrA-DNA complexes have not produced any crystal yet, and further attempts are needed. |
