Nonisotopic assay for DNA repair apurinic/apyrimidinic endonuclease activity using fluorescein isothiocyanate-labeled oligonucleotide substrate and fluorescent image analyzer

IkedaS, Igawa E,  Hayashi Y,  Yamada A, and Tanihigashi H

Apurinic/apyrimidinic (AP) endonuclease plays a pivotal role in repair processing of abasic sites in damaged DNA via the base excision repair pathway. Quantitative assay of AP endonuclease in crude cell extract provides an important aspect for evaluation of the DNA repair potential of the cell. In the present study, we established a nonisotopic assay for measuring incision activity of AP endonuclease using fluorescein isothiocyanate-labeled oligonucleotide substrate and a fluorescent image analyzer. Using this procedure, the incision activity of endogenous AP endonuclease on a synthetic AP site (tetrahydrofuranyl moiety) of an oligonucleotide can be measured in crude extracts from Escherichia coli and Schizosaccharomyces pombe cells. The activity of exogenously expressed human enzyme (hAPE1) in AP endonuclease-deficient bacterial and yeast cells can also be detected

The Bulletin of Okayama University of Science

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