Digestion of chromatin with deoxyribonuclease II

The action of DNAse II on DNA in chromatin was studied. The formation of acid-soluble products followed a two-phase kinetic curve. At the end of the first more rapid phase about 25% of DNA was degraded. Early in the degradation process DNA was converted into double stranded fragments, whose sizes were multiples of about 180 base pairs. As the degradation proceeded these fragments were reduced in size. After denaturation DNA from digested chromatin was resolved into discrete single stranded fractions, exact multiples of a ten-nucleotide length, forming a pattern very similar to that observed with DNAse I.     

Template active chromatin structures: Degradation by deoxyribonuclease I

Chicken embryos were pulse-labelled in vivo with [³H]uridine (10 min), the chromatin isolated and treated with DNAse I. The residual chromatin was separated from the degradation products by centrifugation. The nascent pulse-labelled RNA is completely recovered in the residual chromatin even after prolonged incubation with DNAase I, whereas the DNA is completely degraded to 80 base polynucleotide fragments and smaller fragments.Abbreviations cDNA DNA complementary to mRNA - DOC deoxycholate - EDTA ethylendiamintetraacetic acid - SDS sodium-dodecylsulfate     

Fractionation of native and reconstituted chromatin by digesting with deoxyribonuclease ii

Native and reconstituted chromatin from Ehrlich ascites tumor cells were fractionated into template-active and inactive fractions by the DNAase II/Mg2+-solubility method of Gottesfeld et al. (Gottesfeld, J.M., Garrard, W.T., Bagi, G., Wilson, R.F. and Bonner, J. (1974) Proc. Natl. Acad. Sci. U.S.A. 71, 2193-2197). The Mg2+-soluble (template-active) fractions were compared in respect to sedimentation behavior in sucrose gradients and the relative content of specific transcribed (ribosomal) and non-transcribed (satellite) DNA sequences. It was found that the Mg2+-soluble fraction of the native chromatin was enriched in ribosomal DNA while almost completely devoid of satellite DNA; the nucleoprotein monomer of this fraction sedimented in sucrose gradient at 14 S. Similar-results were obtained if chromatin was fractionated in the presence of 3 M urea. With reconstituted chromatin, however, neither the sedimentation profile, nor the relative content of ribosomal and satellite DNA sequences were recovered, thus indicating that reconstitution did not yield nucleoprotein structurally similar to native chromatin.     

The mechanism of action of the deoxyribonuclease of rat liver chromatin on DNA]

A mechanism of action of DNAase, isolated by chromatin extraction with 0.4 M NaCl, on DNA is described. The enzyme is an endonuclease, it does not require the presence of double-stranded regions in the DNA molecule, does not distinct single-stranded breaks in DNA, and it preferably attacks single-stranded DNA. It hydrolyses DNA for 3'-phosphodiester bonds to octane nucleotides which are resistant to the enzyme activity. The action of the enzyme on DNA does not depend on the position of terminal phosphates. Chromatin DNAase is not specific to DNAs from different origins.     

Release of ribonucleoprotein during digestion of rat testis chromatin with deoxyribonuclease II (3.1.4.6)

The composition of rat testis chromatin proteins in fractions produced by limited DNase II digestion followed by differential precipitation with MgCl2 has been studied. Over 50% of the acid-soluble proteins in the soluble chromatin fraction appeared to be quite similar to proteins which are associated with ribonucleoprotein (RNP) particles in HeLa cells. Although the ratios of the testis RNP protein components differed from those of HeLa RNP particles, the three major polypeptides were most similar to the HeLa components designated A2, B2, and C1. The soluble chromatin fraction was also enriched in the high mobility group proteins HMG1 and HMG2.     

Chromatin sub-structure. The digestion of chromatin DNA at regularly spaced sites by a nuclear deoxyribonuclease

Evidence is presented that indicates a regular sub-structure in nuclear nucleoprotein with regularly distributed sites that are specifically susceptible to the cellular Ca-Mg endonuclease.     

Persistence of the ten-nucleotide repeat in chromatin unfolded in urea, as revealed by digestion with deoxyribonuclease i.

It is shown by enzymatic digestion of chromatin from rat liver or Guerin ascites tumour (GAT) that treatments, which abolish the 180 base pair repeat, as revealed by digestion with micrococcal nuclease (shearing in salt solutions of medium ionic strength, sonication, fixation with formaldehyde in the presence of 5 M urea), have little effect on the 10 nucleotide repeat, observed in deoxyribonuclease I digests.     

Yeast chromatin: Search for histone H1

Summary Yeast chromatin, isolated by a rapid procedure contains in addition to histones H2A, H2B, H3 and H4 a fifth major basic protein. This fifth polypeptide is not an intrinsic component of the nucleosome structure. It has properties of both histone and nonhistone proteins and might represent an early form of histone H1 and of high mobility group nonhistone proteins of higher eukaryotes.Electron microscopic visualization of isolated yeast nucleosomes substantiates further the similarity of the chromatin structure of this unicellular eukaryote to that of higher eukaryotes.     

Rat liver chromatin digested in situ with endogeneous Mg(2+)-dependent deoxyribonuclease exhibits two distinct DNA repeats.

Incubation of rat liver nuclei in the presence of 1.0-5.0 mM Mg2+ at 37 degrees C releases oligonucleosomes containing at least two distinct chromatin-DNA repeat elements. The 'short' repeat is derived from the dimer to pentamer series, while the 'long' repeat is found in the monomer and hexamer to decanucleosomes. Both repeat lengths decrease during enzymatic hydrolysis but in 5.0 mM Mg2+, which is optimal concentration, the 'long' repeat is degraded faster.     

Human genetically polymorphic deoxyribonuclease: purification, characterization, and multiplicity of urine deoxyribonuclease I

A deoxyribonuclease I was purified from the urine of a 46-year-old male (a single individual) by using a series of column chromatographies to a homogeneous state as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was found to be a glycoprotein, containing 1 fucose, 7 galactose, 10 mannose, 6 glucosamine, and 2 sialic acid residues per molecule. The N-terminal amino acid sequence up to the 27th residue of the enzyme was similar to that of pancreatic deoxyribonuclease I from bovine and other species. The catalytic properties of the enzyme derived from a single individual closely resembled those of deoxyribonuclease I purified from human urine collected from several volunteers [Ito, K. et al. (1984) J. Biochem. 95, 1399-1406]. The purified enzyme was found to consist of multiple forms with different pI values. These findings are compatible with the existence of genetic polymorphism of deoxyribonuclease I in human urine previously reported [Kishi, K. et al. (1989) Hum. Genet. 81, 295-297]. This multiplicity of the urine enzyme might be due to variations in the primary structure and/or differences in the content of sialic acid.     

Human urine deoxyribonuclease increases endogenous thymidine in the mouse thymocyte interleukin 1 assay: an artificial interleukin-1 inhibitor

Molecular size chromatography of urine from normal individuals showed two peaks of apparent IL-1 suppressive activities when tested by the murine thymocyte comitogenic assay (mol wt greater than 600 kD and 20 to 60 kD). However, the high molecular weight inhibitory activity disappeared if the concentration of PHA was increased during assay, and the low molecular weight inhibitory activity subsided in the presence of a high concentration of [3H]thymidine. The 20 to 60 kD fractions contained DNase activity which acted on DNA liberated from the considerable number of dying thymocytes during the course of the assay. Thus, incubating the urine fractions with freeze-killed murine T cells, whose DNA was prelabeled with [3H]thymidine, showed the appearance of supernatant [3H]thymidine correlating quantitatively with the DNase activity in the fractions. This indicates that urine DNase together with phosphatase(s) in the thymocyte cultures increase the level of extracellular, unlabeled thymidine, thereby diluting the specific activity of the tracer. These artificial IL-1-inhibitors may explain why urine from both normal and febrile individuals 'inhibits' IL-1 only when tested for thymocyte-activating activity but not when tested for other biological activities.     

Cdt1 associates dynamically with chromatin throughout G1 and recruits Geminin onto chromatin

To maintain genome integrity, eukaryotic cells initiate DNA replication once per cell cycle after assembling prereplicative complexes (preRCs) on chromatin at the end of mitosis and during G1. In S phase, preRCs are disassembled, precluding initiation of another round of replication. Cdt1 is a key member of the preRC and its correct regulation via proteolysis and by its inhibitor Geminin is essential to prevent premature re-replication. Using quantitative fluorescence microscopy, we study the interactions of Cdt1 with chromatin and Geminin in living cells. We find that Cdt1 exhibits dynamic interactions with chromatin throughout G1 phase and that the protein domains responsible for chromatin and Geminin interactions are separable. Contrary to existing in vitro data, we show that Cdt1 simultaneously binds Geminin and chromatin in vivo, thereby recruiting Geminin onto chromatin. We propose that dynamic Cdt1-chromatin associations and the recruitment of Geminin to chromatin provide spatio-temporal control of the licensing process.