To live, humans convert oxygen to energy. During this process, metabolic byproducts are formed known as reactive oxygen species (also known as free radicals). These reactive products attack cellular constituents, such as lipids, proteins and DNA. Reactions with DNA, i.e. our genetic material, can lead to several damage intermediates. If unrepaired, this damage can promote unwanted genetic change or lead to cell death. Such end-points are associated with cancer, neurodegeneration, and the aging process. To regulate these outcomes, organisms have evolved an array of repair systems, which recognize and remove specific forms of DNA damage. Base excision repair (BER) is the major pathway for repairing oxidative DNA damage and involves the cooperative interaction of several proteins that work sequentially to excise the target damage and restore DNA back to its original, unmodified form. Our focus has been to understand the molecular mechanisms of repair of abasic sites and oxidative DNA single strand breaks. Towards this end, we have isolated several BER protein participants and are defining their individual and cooperative structure-function relationships. Our studies have revealed that Ape1, a central participant in BER and the major mammalian repair protein for AP sites, is a structure-specific endonuclease that scans DNA for a unique flexibility associated with the abasic lesion. We have defined how this enzyme cuts DNA - the first step in abasic site repair - a catalytic reaction mechanism that is likely conserved throughout evolution by a superfamily of enzymes. While Ape1 operates as the predominant (if not only) mammalian enzyme in AP site repair, we have shown that it has a more targeted role in the excision of 3'-blocking (e.g. phosphate) damages, depending on DNA context/structure; thus other proteins likely contribute to this corrective process. Moreover, we have shown that Ape1 is an editing factor that removes certain 3'-terminal mismatched nucleotides, potentially mutagenic DNA intermediates. We are presently determining the mechanism by which Ape1 communicates with other proteins in the BER pathway, most notably DNA polymerase beta, using biochemical, NMR spectroscopy and crystallography techniques. Our structure-function analysis is being expanded into defining the biochemical and cellular functions of Xrcc1, a protein that has been proposed to operate as a scaffolding factor in BER by binding DNA nicks and gaps, and recruiting BER proteins. Additional investigation, which involves the use of clinical samples from the BLSA, includes understanding the impact of genetic variation found in the human population on DNA repair function, with the hypothesis that certain genetic differences produce proteins that are less effective at DNA repair, thus rendering the individual more susceptible to disease upon exposure to environmental or food agents that create oxidative damage. In summary, by understanding the basic operations of DNA repair, we are building a foundation upon which we can better understand the relationship of genetic variation in oxidative DNA damage response systems to human disease and the aging process.
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