The action of pancreas deoxyribonuclease I (deoxyribonucleate oligonucleotidohydrolase, EC-number 3.1.4.5.) on calf thymus nucleohistone

The DNA-components of thymus nucleohistone 2 (TNH) are hydrolyzed by pancreas deoxyribonuclease I in the presence of Mg 2+-ions to mixtures of acid-soluble oligonucleotides and of cleavage nucleoproteins (c-NPs) 2 2 Abbreviations. Thymus nucleohistone, TNH; cleavage nucleoproteins, c-NPs; deoxyribonuclease, DNase. which contain the total amount of the acid-insoluble portion of the hydrolyzed DNA-components and almost the total amounts of the substrate proteins. The kinetics of this hydrolysis is characterized by a rapid initial phase up to hydrolysis degrees 3 3 The term “degree of hydrolysis,” as used in this investigation for the extent of the enzymatic cleavage of DNA-chains into smaller fragments, is defined as the ratio acid-soluble P formed: total P of the substrate. of approximately 50% which is followed by a much slower continuation of the enzymatic hydrolysis. During this phase, the c-NPs precipitate almost quantitatively. In contrast to the anionic nature of the substrate-TNH, in which the cationic groups amount to only 57%, of the total anionic groups, the corresponding ratio of the insoluble c-NPs is stoichiometric. The decrease of the phosphoryl groups of the nucleoproteins during the incubation with DNase I is accompanied by corresponding decreases of their magnesium-binding capacities. The rapid initial and the sluggish second phase of the DNase I-catalyzed hydrolysis of TNH was observed not only with dissolved substrate-TNH, but also with suspensions of the insoluble complexes of TNH with stoichiometric amounts of Mg 2+-ions or of diamines of low molecular weights. This made it unlikely that the sluggishness of the hydrolysis during the second phase could be explained as a consequence of the coarse dispersion of the insoluble c-NPs. On the other hand, homogenized suspensions of the insoluble c-NPs or of insoluble, stoichiometric complexes of TNH with polylysines were hydrolyzed by DNase I at much slower initial rates than those observed with the TNH-magnesium or the TNH-diamine-complexes. This suggests that the initial rapid phase of the DNase I-catalyzed cleavage of TNH involves predominantly those segments of the substrate-DNA-chains that are not directly bound to the cationic amino acid residues of its histone components. The mixture of the acid-insoluble DNA-components of the c-NPs (they account for 100% of the total P of the soluble c-NPs, but only for 75–85% of the total P of the insoluble c-NPs) was sedimented in a sucrose density gr