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Chair(s): Dedon, Peter
(SY064) Ape1, DNA polymerase-beta and the repair of oxidized abasic sites: Roadblocks, detours and divided highways.
Demple, Bruce*,1, 1 Dept. of Genetics & Complex Diseases, School of Public Health, Boston, MA, USA
ABSTRACT- Deoxyribose is an important target for free radical damage within DNA, which generates various types of oxidized abasic sites, and their repair would be important for cell viability and the maintenance of genetic stability. We have studied the role of mammalian base excision repair (BER) pathways in correcting these lesions. The central BER players include Ape1 protein and DNA polymerase beta. Ape1 has been well characterized as an apurinic (AP) endonuclease, but the enzyme very effectively cleaves at the 4'-oxidation product 2-deoxypentos-4-ulose and the 1'-oxidation product 2-deoxyribonolactone (dL). Ape1 is also the major activity in human cells for removing fragments such as phosphoglycolate esters from 3'-termini at oxidative strand breaks. The 2-deoxypentos-4-ulose residues cleaved by Ape1 are effectively removed in a beta-elimination reaction catalyzed by DNA polymerase beta. The dL residues, however, present a special problem: following rapid Ape1 cleavage, attempted excision by polymerase beta leads to stable protein-DNA crosslinks (linked via amide bonds) that block further BER. We have explored the mechanisms by which mammalian cells avoid or repair these crosslinks, and mobilize alternative pathways to complete dL repair. We have also explored the fundamental role of Ape1 using RNA interference techniques to suppress the protein in human and mouse cells. Suppression of Ape1 levels below about 30% of normal leads to the arrest of cell proliferation, the accumulation of unrepaired abasic DNA damage formed spontaneously, and the activation of apoptosis. At earlier times or lesser suppression levels, the role of Ape1 in dealing with DNA damage can be assessed. In this way, we have shown that Ape1 handles oxidative damage formed by hydrogen peroxide or ionizing radiation. The role of DNA repair in all these effects was demonstrated by supplying an alternative, unrelated AP endonuclease (yeast Apn1).
Key words: abasic sites, oxidative DNA damage, base excision DNA repair, apoptosis
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