Genetic material is subject to spontaneous and mutagen-induced modifications that contribute to the process of carcinogenesis. The most frequently formed damages are likely those produced by free radical attack of DNA. Free radicals, which include superoxide and hydroxyl radicals, are generated during normal oxygen metabolism and from exposure to anti-cancer agents such as bleomycin and ionizing radiation. Mutagenic or lethal oxidative DNA damages include sites of base loss (abasic or AP sites) and 3'-obstructive termini (3'-phosphate or 3'- phosphoglycolate). Enzymes that initiate the repair of AP sites and 3'- damages have been identified in all organisms studied to date, from bacteria to humans, and are a central component of base excision DNA repair. The predominant AP endonuclease in mammals is Ape/Hap1/Ref-1, but unlike other AP endonucleases, Ape possesses only a minor 3'-repair activity. Although many of the general features of Ape have been elucidated, the specifics of its recognition and binding to abasic site-containing DNA are not well understood. Using biochemical (e.g. footprinting, protein-DNA crosslinking, and site-directed mutagenesis) and structural (Nuclear Magnetic Resonance and Circular Dichroism) approaches, we intend to unravel the details of the Ape-DNA interaction and to identify the critical points of contact. In addition, we will employ computer modeling to define the structural features of DNA substrates utilized in these and other studies to uncover aspects of DNA that are important for recognition and incision. Elucidating the molecular detail of the Ape-DNA interaction may permit the design of effective inhibitor substrates, which may aid cancer treatment regimes and Ape-DNA structural studies, or of a dominant-negative Ape protein, which would help to clarify the biological importance of this enzyme. Since Ape displays a relatively poor 3'-diesterase activity in vitro, particularly at double-strand break ends, the biological role of this activity is unclear. A recent study reporting an interaction between Ape and Ku could suggest that these proteins act in cooperation to efficiently process 3'-damages, and may indicate a role for Ape in non- homologous recombination (i.e. DNA end-joining). We intend to characterize this interaction biochemically and determine whether Ku influences the 3'-repair capacity of Ape. We intend to identify and characterize other nucleases or accessory factors that are involved in the processing of 3'-oxidative damages. The studies described within will go a long way towards defining the repair activities of Ape and will help understand the intracacies of two critical DNA repair pathways.
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