Methods for limiting the action of SP3 DNAase and for the determination of the direction of hydrolysis of processive exonucleases.
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.
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
D009838:Oligodeoxyribonucleotides; D004247:DNA; D003851:Deoxyribonucleases; D005092:Exonucleases