Studies of the Specificity of Deoxyribonuclease I III. HYDROLYSIS OF CHAINS CARRYING A MONOESTERIFIED PHOSPHATE ON CARBON 5

Kunitz (3) crystallized pancreatic deoxyribonucleasel and studied its kinetics and the products of its action on deoxyribonucleic acid. With a ratio of enzyme to substrate not higher than 1: 500, the products obtained after exhaustive digestion had an average length of a tetranucleotide. Studies from this and Sinsheimer’s laboratory (for details see the recent review (4)), showed that the digest thus obtained represented a complex mixture of fragments varying in length from mononucleotides to at least o&a-, possibly dodecanucleotides. The attempts to redigest larger oligonucleotides (by the use of a low enzyme-substrate ratio) into smaller fragments failed (5). It was therefore assumed that the digest thus obtained is a “limit digest” and that it is composed of fragments containing only internucleotide linkages which are totally resistant to the action of DNase I. Hurst (6) and Hurst and Fmdlay (7) observed that with a very large amount of enzyme (ratio of enzyme to substrate of the order 1: lo), at pH 8.0, in the presence of ethylenediaminetetraacetate and Mn++, the hydrolysis proceeds beyond the “limit digest” stage. To explain their findings, Hurst and Findlay (7) postulated that crystalline DNase I contains still another enzyme, an oligonucleotidase, which is capable of hydrolyzing oligonucleotides present in the “liiit digest” into smaller fragments, as measured by solubility in a uranyl acetate reagent. Independently, in the previous paper of this series (2), it was observed that when oligonucleotides carrying monoesterified phosphate on carbon 3’ were digested with very high amounts of DNase I, the predominant products were derivatives of dinucleotides. The hypothesis was advanced (2) that dinucleotides represent an ultimate product of the action of DNase I on deoxyribonucleic acid and that an apparent end point (“limit digest”) is caused by an insufficient amount of enzyme and by product inhibition. Thus, an alternative explanation was suggested, without postulating