Date of Award

1985

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Abstract

The work presented herein investigates the repair of apurinic/apyrimidinic (AP) sites in Drosophila melanogaster. Two different AP endonucleases were isolated differing in pH optima, reaction requirements, and associated activities. AP endonuclease I flows through phosphocellulose, has a pH optimum of 6.5, and has no obvious cofactor requirements for the incision of AP DNA. AP endonuclease I additionally has associated non-specific endonuclease activity, inseparable throughout the purification. AP endonuclease II is retained by phosphocellulose, eluting between 200-400 mM potassium phosphate. This enzyme has an absolute requirement for magnesium, a pH optimum of 7.0, and no detectable associated enzyme activities. Both enzymes cleave the phosphodiester backbone of AP DNA, with no activity on alkylated, heat-denatured, UV, or OsO(,4)-treated DNA substrates. Upon further purification of the enzymes, Sephadex G-100 analysis gave molecular weight estimates of 65-70,000 daltons. Moreover, the two D. melanogaster AP endonucleases and E. coli endonuclease IV were found to cross-react with an antibody prepared against a human AP endonuclease, pointing to conservation of some antigenic determinants through evolution. Using this cross-reactivity, immunoblots of SDS-PAGE gave estimates of 68,000 daltons for AP endonuclease I and 64,000 daltons for AP endonuclease II. The precise incision position relative to the AP site was also determined for each D. melanogaster endonuclease. AP endonuclease I cleaves DNA 3' to the AP site, producing a 3'-deoxyribose-P and a 5'-OH. AP endonuclease II also nicks DNA 3' to the AP site, but leaves a 3'-deoxyribose-OH and a 5'-P terminus. In the context of DNA repair in general, the results presented here provide the first example of non-complementary classes of AP endonucleases and additionally identifies the first AP endonuclease that leaves a 3'-P terminus. The data presented will hopefully open the way for analysis of the mutagen sensitive D. melanogaster stocks. Toward the end, data is presented on AP DNA repair in two D. melanogaster strains deficient for base excision repair. Analysis of these mutagen sensitive strains should lead to a better understanding of the role of AP endonucleases in eukaryotic DNA repair.

Pages

106

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