Oxidative stress is arguably the most important cause of cellular genotoxicity. The reactive oxygen species (ROS), generated spontaneously due to oxidative stress, and exogenously by ionizing radiation (IR) and other environmental agents, are the major cause of various mutagenic, carcinogenic, and cytotoxic lesions in DNA that are implicated in the etiology of a variety of pathophysiological disorders including aging and cancer. The oxidative DNA damage includes single and double strand breaks and a battery of base damage. These oxidative base lesions are generally repaired via the base excision repair (BER) pathway in bacteria and eukaryotes, initiated by removal of the damaged base by a damage-specific DNA glycosylase or glycosylase/lyase. Among such oxidatively damaged bases, a series of toxic and mutagenic structurally diverse oxidized pyrimidines are repaired by endonuclease III (NTH), a glycosylase/lyase present in all species from bacteria through man. Little is known about its detailed mechanisms either for recognition of substrates of various structures or for catalysis, and also about additional cellular factors that interact with NTH or other key factors involved in NTH-initiated BER, and which control the length of the repair patch to facilitate efficient in vivo repair. We have recently cloned the human endonuclease III (hNTH1) cDNA, and purified the active recombinant protein in large quantity from E. coli. We now propose to test the hypothesis that the highly basic hNTH1 enzyme binds DNA, scans along it by facilitated diffusion, recognizes oxidized pyrimidines, then locks the flipped-out base in its catalytic pocket, and finally attacks the C-1 of sugar to cleave the glycosyl bond. We will also test the hypothesis that for efficient in vivo repair, hNTH1 interacts with other cellular factors that determine the length of the repair patch size which distinguishes the BERI (single nucleotide incorporation) vs. BERII (multiple nucleotides incorporation). The objectives of this project are to elucidate the role, structure-function relationship, and reaction mechanisms of hNTH1.
Our specific aims are: (1) to characterize the molecular mechanisms of substrate recognition and catalysis by hNTH1; (2) to determine whether hNTH1-mediated oxidative DNA damage repair belongs to BERI or BERII pathway; (3) to characterize possible interaction of hNTH1 with cellular factors including other BER proteins. These studies will use various standard molecular biology and biochemical techniques, as well as fluorescence spectroscopy. Our long-term goal is a comprehensive understanding of the role and regulation of NTH as a component of mammalian BER system for repair of cellular oxidative damage. This knowledge will allow us eventually to devise strategies for modulating NTH expression for chemopreventive and therapeutic purposes.
