Repair Mechanisms For Strand Breaks in DNA

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To live, humans convert oxygen to energy. Yet during this process, metabolic byproducts are formed known collectively as reactive oxygen species (also known as free radicals). These reactive products attack various cellular constituents, including 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 human disease, most notably cancer and neurodegeneration, and to 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 the DNA back to its original, unmodified form. In brief, the main steps of BER consist of: (1) excision of the damaged base (e.g. 8-oxoguanine), (2) incision of the DNA backbone at the abasic site product, (3) removal of the abasic terminal fragment, (4) gap-filling synthesis, and (5) ligation of the final nick. Our focus has been to understand the molecular mechanisms of BER for two common oxidative DNA damage intermediates, specifically abasic sites and DNA strand breaks that harbor non-conventional 3?-blocking termini (e.g. phosphates). 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 abasic sites, is a structure-specific endonuclease that scans DNA for a unique flexibility associated with the abasic lesion. While this protein operates as the predominant (if not only) mammalian enzyme in abasic site repair, we have shown that it has a more limited role in the excision of 3?-blocking damages, depending on DNA context/structure; thus other proteins likely contribute to this corrective process. Presently, we are determining the mechanism by which Ape1 cuts DNA (the first step in removing the abasic damage) and communicates with other proteins in the BER pathway, most notably DNA polymerase beta and Xrcc1, using biochemical, NMR spectroscopy and crystallography techniques. Our structure-function analysis of proteins in BER is now being expanded into understanding the impact of genetic variation found in the human population on DNA repair function. The hypothesis is that certain genetic differences will produce proteins that are less effective at DNA repair, thus rendering the affected individual more susceptible to environmental or food agent exposures that induce oxidative stress and increase oxidative damage. We have recently shown that indeed genetic differences in APE1 can lead to proteins with reduced repair efficiency. 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.