Mechanism of action of micrococcal nuclease on deoxyribonucleic acid.

Two types of methods have been used for the elucidation of specificity (or preference) of nucleases when complex polynucleotides are used as substrates. One method depends on the identification of products in the digest. I f a particular combination (or combinations) is missing, it means that this combination is preferentially attacked. Thus, in the case of pancreatic ribonuclease, sequences Pyp-Pyp,’ Pyp-Pup are absent in the digest, therefore, the bond specifically cleaved is Pyp-Xp, where X is either purine or pyrimidine. A second method of studying specificity is to isolate oligonucleotides and to determine the frequency of nucleosides occurring at each end of the chain. This can be accomplished by several means. The most general method is to remove the terminal monophosphate by prostatic phosphatase, and then degrade an aliquot with venom phosphodiesterase and another aliquot with spleen phosphodiesterase. The nucleoside obtained by the degradation with venom phosphodiesterase indicates the 5’-terminus, and that with spleen phosphodiesterase, the 3’-terminus. A modification of this method, applicable only to oligonucleotides bearing 3’.monophosphate, is to degrade with venom phosphodiesterase directly. In this case the 5’.hydroxyl terminus appears as a nucleoside, and the 3’-phosphoryl terminus as a nucleoside 3’) 5’-diphosphate. The validity of these methods in elucidating the specificity of a nuclease depends on the tacit assumption that specificity remains the same throughout the entire course of the reaction. An observation (2) that the proximity of monoesterified phosphate exerts a pronounced influence on the susceptibility of neighboring linkages to deoxyribonuclease I led us to believe that this assumption is false. With several nucleases (Z-5)) the cleavages that occur in the early part of the reaction are not identical with those occurring later. The second factor responsible for the changing behavior is the gradation of preference. Rushizky, Knight, and Sober (6) have recently shown that pan-