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

Abstract 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 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 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 gradient. Only one peak was obtained which coincided with that of a reference sample of yeast tRNA. The sedimentation pattern of the DNA-component of the c-NPs did not significantly change between hydrolysis degrees of 17 and 60%, except for the fact that the yields of the acid-insoluble polynucleotide fraction of the c-NPs decreased appreciably during the later phases of the enzymatic digestion owing to the slow degradation of the c-NPs. With regard to the distribution of the histone components of the substrate-TNH along its DNA-chains, it seems that “histone-bound” 4 DNA-segments of mutually similar chain lengths alternate with “histone-free” 4 segments of unknown individual chain lengths. The resistance of insoluble TNH-complexes with spermidine and spermine, respectively, against the action of DNase I was found to be unexpectedly high in view of the low molecular weights of these compounds and of the small number of cationic groups per molecule. These observations suggest the important role of structural features such as the chain lengths of the alkyl bridges connecting the pairs of amine groups for the stability of the complexes of a cationic polyelectrolyte with TNH.