Biophysical studies of O(6)-alkylguanine-DNA alkyltransferase

O6-Alkylguanine-DNA alkyltransferase (AGT) is a monomeric DNA repair protein whose homologs are found in a variety of organisms. Unlike many proteins responsible for maintenance of genomic integrity, AGT is not an enzyme, but restores DNA by irreversible transfer of O6-guanine adduct substituents to an active-site sulfur atom. The protein prevents tumorigenesis in normal tissues exposed to alkylating agents, but also confers chemotherapeutic resistance to cancer cells.; A recent crystal structure of human AGT (hAGT) suggests the presence of a previously unrecognized zinc atom bound within the N-terminus. Investigation of the effects of zinc on hAGT represents one major focus of this dissertation. Zinc enhances the protein's expression, and higher occupancy of the novel metal binding site correlates with faster repair rates. Mutations of the residues involved in zinc binding diminish metal occupancy and reduce repair efficiency without inducing major structural changes in hAGT. Zinc binding is correlated with greater protein stability as evidenced by resistance to urea denaturation. Based upon these results, it is postulated that zinc occupancy may affect the DNA repair process in vivo and may also be involved in regulation of hAGT.; Given the nonenzymatic nature of the protein, protection afforded by AGT is likely to depend upon regulation of its synthesis and degradation, and upon its ability to efficiently locate repairable lesions throughout the genome. AGT-DNA interaction is central to this process, and it represents the other major area of investigation detailed in this thesis. Biophysical experiments show that while hAGT sediments as a monomer in the absence of DNA, it binds cooperatively to single-stranded and double-stranded oligodeoxyribonucleotides. This strong cooperative interaction and the corresponding stoichiometry of complex formation are unperturbed by active-site mutation or by alkylation of either AGT or DNA. In this binding mode, AGT discriminates poorly between lesion-containing and lesion-free DNA. These findings support a novel model of lesion search and recognition that involves cooperative formation and processive movement of multiprotein complexes.; The biophysical aspects of DNA repair by hAGT elucidated herein may influence the success of clinical protocols designed for both the prevention and treatment of neoplastic disease.