title: Methods for limiting the action of SP3 DNAase and for the determination of the direction of hydrolysis of processive exonucleases.
abstract: The action of the exonuclease SP3 DNAase is inhibited by chemical modification of DNA with the cation N-cyclohexyl-N'-beta-(4-methylmorpholinium)-ethylcarbodiimide (CME). The limited activity of the enzyme on CMA-modified DNA makes it possible to demonstrate that the enzyme also initiates its attack on polydeoxyribonucleotides at the 5'-termini. This was determined by the analysis of the products from the digestion of CME-modified DNA containing labeled 5'-terminal phosphate groups. Such procedure can be adopted as a general approach for the determination of the direction of hydrolysis of other processive exonucleases. SP3 DNAase has been shown able to degrade oligo- and polydeoxyribonucleotides with or without 5'-terminal phosphate groups with equal efficiency (Aposhian, H.V., Friedman, N., Nichihara M., Heimer, E.P., and Nussbaum, A.L. (1970) J. Mol. Biol. 49, 367-379). The present work also shows that the enzyme can even hydrolyze oligo- and polynucleotides containing derivatized phosphate groups.
mesh_terms: D001412:Bacillus subtilis / Q000201:enzymology; D001435:Bacteriophages / Q000201:enzymology; D004247:DNA; D003851:Deoxyribonucleases / Q000378:metabolism*; D005092:Exonucleases / Q000378:metabolism*; D007700:Kinetics; D009838:Oligodeoxyribonucleotides / Q000032:analysis
chemical_list: D009838:Oligodeoxyribonucleotides; D004247:DNA; D003851:Deoxyribonucleases; D005092:Exonucleases