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A single apurinic site can elicit BAL 31 nuclease-catalyzed cleavage in duplex DNA☆

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Abstract

The extracellular nuclease from Alteromonas espejiana BAL 31 is a highly sensitive endonucleolytic probe for lesions that distort the helical structure of duplex DNA. The nuclease can be isolated as two distinct molecular species, the ‘fast’ (F) and ‘slow’ (S) species, which have different kinetic properties. When nonsupercoiled, covalently closed circular phage PM2 DNA containing apurinic sites introduced by heating at acid pH was incubated with individual fractions from a chromatographic column which separated the two nuclease species, cleavage of the DNA was observed which was greatly in excess of control levels using nonmodified DNA. The initial rates of such cleavage clearly paralleled the peaks of both absorbance and nuclease activity against single-stranded and linear duplex substrates. When samples of apurinic DNA were incubated with pooled fractions from the same column representing pure F and S nucleases, respectively, the rate and extent of the cleavage observed was dependent upon the average number of apurinic sites per molecule. Cleavage was readily detectable in samples containing an average of 1.1 apurinic sites per molecule with both species of the enzyme. These results indicate that either species of the BAL 31 nuclease can recognize and cleave in response to a single apurinic site in duplex DNA. The F nuclease appears to be approx. 2.5-times as efficient in cleaving DNA containing apurinic lesions as the S enzyme in extended incubations.

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☆

This is paper VI in the series ‘Extracellular Nucleases of Alteromonas espejiana BAL 31.’ Paper V is Ref. 5.

∗∗

Present address: Department of Cell Biology, Baylor College of Medicine, Houston, TX 77030, U.S.A.

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Copyright © 1984 Published by Elsevier B.V.