Deoxyribonuclease II as a probe for chromatin structure: II. Mode of cleavage

Abstract

In the analysis of DNAase II digestion of chromatin, as described in the preceding paper, interactions between adjacent nucleosomes play an important part. In order to understand the mechanism of DNAase II cleavage we next investigated the role of histone H1 in these interactions and characterized the nucleoprotein particles arising in the course of DNAase II action. H1-free chromatin prepared by three different procedures, using either 0.6 m-NaCl, transfer RNA or an ion-exchange resin, can be cleaved by DNAase II only at the internucleosomal cleavage site leading to 200-bp † † Abbreviations used: bp, base-pairs; PMSF, phenylmethanesulfonyl fluoride; EGTA, ethyleneglycol bis(β-aminoethyl ether)- N,N′-tetraacetic acid. digestion patterns regardless of the ionic conditions. When H1 was added back to the three chromatin preparations the 100-bp cleavage pattern could be restored only with material prepared by the resin method at low concentrations of salt. Addition of polylysine instead of H1 has the same effect, but only with material prepared by that method. A direct correlation between extended and condensed states of chromatin as monitored by electron microscopy and DNAase II cleavage in the 200 and 100-bp modes, respectively, could be established. The continuity of the nucleosome chains in DNAase II-digested chromatin is maintained in spite of intranucleosomal cleavage in the terminal section of the core DNA, even in the absence of H1. Addition of 3 m-urea, however, disrupts the nucleosome chains at the intranucleosomal cleavage sites and leads to the formation of novel nucleoprotein particles as seen in sucrose gradient centrifugations. Those sedimenting between mononucleosomes and dinucleosomes contain, almost exclusively, DNA of 300 bp (mouse) or 315 bp (chicken erythrocyte). They can be formed from particles sedimenting in the absence of urea in the dinucleosome region by either a dissociation process or a massive conformational change. On the basis of the results presented here and in the preceding paper a mechanism for DNAase II cleavage of chromatin in the 200-bp and 100-bp modes is proposed and discussed in the context of structural features of chromatin recognized by DNAase II.