Sea urchin sperm DNP. I. Chemical composition and template properties of DNP

Electrophoretic mobility, amino acid composition and salt dissociation of histones isolated from sperm of sea urchin Strongylocentrotus intermedius and calf thymus cells were studied. The special arginine-rich histone fraction (I) has been observed in sea urchin sperm chromatin, this fraction being absent in calf thymus chromatin. Dissociation of lysine-containing histone fractions from sea urchin chromatin occured in the range of 0.7 to 1.0 M NaCl concentrations. H1 of calf thymus chromatin was totally extracted with 0.6 M NaCl. In the course of a further increase of salt concentrations (up to 1.5 M NaCl) a practically total extraction of histones from sperm chromatin was observed, while about 20% of proteins remained bound to DNA in thymus chromatin after extraction with 2.0 M NaCl. The template activity of non-extracted DNP preparations from urchin sperm was equal to 2-3% of that of totally deproteinized DNA. The template activity of DNP gradually increased at protein extraction from DNP preparations. The hybridization capacity of RNA transcribed on partially dehistonized DNP templates in vitro also increased.     

Specific interaction of histone H1 with eukaryotic DNA.

The interaction of calf thymus histone H1 with homologous and heterologous DNA has been studied at different ionic strengths. It has been found that about 0.5 M NaCl histone H1, and its fragments N-H1 (residues 1-72) and C-H1 (residues 73-C terminal), precipitate selectively a small fraction of calf thymus DNA. This selective precipitation is preserved up to very high values (less than 2.0) of the input histone H1/DNA ratio. The percentage of DNA insolubilized by histone H1 under these ionic conditions is dependent upon the molecular weight of the nucleic acid, diminishing from 18% fro a Mw equals 1.0 x 10(7) daltons to 5% for a Mw equals 8.0 x 10(4) daltons. The base composition of the precipitated DNA is similar to that of the bulk DNA. Calf thymus histone H1 also selectively precipitates a fraction of DNA from other eukaryotes (herring, trout), but not from some prokaryotes (E. coli, phage gamma. On the other hand, at 0.5 M NaCl, the whole calf thymus DNA (but not E. coli DNA) presents a limited number of binding sites for histone H1, the saturation ratio histone H1 bound/total DNA being similar to that found in chromatin. A similar behavior is observed from the histone H1 fragments, N-H1 and C-H1, which bind to DNA in complementary saturation ratios. It is suggested that in eukaryotic organisms histone H1 molecules maintain specific interactions with certain DNA sequences. A fraction of such specific complexes could act as nucleation points for the high-order levels of chromatin organization.     

Acidic pentapeptide phosphorylated in vitro by calf thymus protein kinase NII binds to DNA in the presence of Mg2+ cations

The pentapeptide pyroGlu-Ala-Glu-Ser-Asn has been synthetized and phosphorylated in vitro at level of serine by protein kinase NII isolated from calf thymus chromatin. It is noteworthy that the calf thymus kinase NII shows a remarkable affinity for this peptide. The [32P]peptide is able to bind to several DNAs in the presence of Mg2+ (lambda phage, calf thymus, pBR540 plasmid). This binding appears not specific with regard to the type of DNA and its base sequence. These data support the hypothesis that phosphorylated acidic domains of nuclear nonhistone proteins could bind directly to DNA in the presence of Mg2+ cations.     

Differential scanning calorimetry of chromatin at different levels of condensation

The thermal denaturation of calf thymus total chromatin and of fractions enriched in heterochromatin or euchromatin, has been investigated by differential scanning calorimetry and compared to that of calf thymus DNA and DNA-histone complexes. In our experimental conditions, chromatin melts in three thermal transitions: the main one, assigned to separation of the DNA double helix, occurs at 83 °C, while the other two occur at 63 °C and 74 °C. The data show that: (a) the transition enthalpy for denaturation of DNA in the total chromatin and in DNA-histone complexes is nearly the same as that of DNA in solution; (b) the transition at 63 °C is present in the thermogram of the heterocromatin enriched fraction, while it is completely absent in that of the euchromatin enriched one. The results suggest that this transition can be attributed to the higher order structures of heterochromatin.     

The role of H2B histones from the sea urchin sperm in the formation of supranucleosome structures

The structural role of histone H2B from sea urchin sperm (H2Bsp) has been examined in experiments on reconstitution of chromatin from DNA and core histones taken in three variants: (1) four core histones from sea urchin sperm; (2) four core histones from calf thymus; (3) (H3, H4, H2A) from calf thymus and H2Bsp. It is shown that H2Bsp when present in reconstituted chromatin induces its aggregation. Fidelity of the reconstitution of nucleosomes has been tested using DNase I probe, one- and two-dimensional electrophoresis and electron microscopy. The reconstitutes that contain H2Bsp appear under electron microscope mainly as regular closely spaced large granules, about 450 A in diameter, which are very similar to the granules found in "native" sea urchin sperm chromatin. The reconstitutes formed by four core histones from calf thymus appear as randomly arranged particles, about 100 A in diameter. We conclude that histone H2Bsp participates in interactions between nucleosomes and is involved in the formation of the condensed supranucleosomal structure in sea urchin sperm chromatin.     

Bovine thymus poly(adenosine diphosphate ribose) polymerase.

About 1,300-fold purification of poly(adenosine diphosphate ribose) polymerase has been achieved from the extract of bovine thymus with a recovery of 10 to 20%. The final preparation has a purity of 99%, and the enzyme is composed of a single peptide with a molecular weight of 130,000. The purified enzyme required NAD+, Mg2+, a thiol compound, DNA, and histones for full activity. Whereas DNA is essential for activation of the enzyme, histones are not. The observed stimulation of the reaction by histones is shown to be due to masking of the inhibitory effect of contaminating denartured DNA in native DNA preparation. The concentration of DNA required for half-maximal enzyme activity (apparent Km for DNA) is proportional to the concentration of enzyme in the reaction mixture. The minimum estimation of the number of nucleotide pairs of DNA required for half-maximal activation of one enzyme molecule is 220 to 240 for bulk of calf thymus DNA, while the value is 10 for a calf thymus DNA fraction, "active DNA," which was separated from the enzyme fraction in a stage of the purification. These results suggest that the enzyme is activated by binding to a specific site on calf thymus DNA. The apparent Km for NAD+ and the maximum velocity of the enzyme are estimated to be 60 micrometer and 0.91 mumolper min per mg, respectively.     

Visualization of poly(ADP-ribose) synthetase associated with polynucleosomes by immunoelectron microscopy

Antibodies showing a high specificity for poly(ADP ribose) synthetase have been purified. A fraction binding nonspecifically to histones present in antiserum and non-immune serum has been demonstrated by immunoblotting and separated by histone-Sepharose chromatography. The antibody without the nonspecific binding fraction was analyzed by Western blot with calf thymus protein extract and was found to react only with a band at 116 kDa. There was no reaction with purified topoisomerase I, this weak activity was copurified with poly(ADP-ribose) synthetase preparation. The specific IgG fraction has been used for the visualization of the interaction of poly(ADP-ribose) synthetase with chromatin by indirect gold-labelling. This immunomicroscopic study suggests that the synthetase is located in the inner part of polynucleosomes and would be associated preferentially with the core nucleosome.     

Electric dichroism of chromatin

The linear dichroism of sheared calf thymus chromatin, oriented in solution by a pulsed electric field, has been measured. The limiting value of this dichroism is considerably less negative than that of calf thymus DNA, but does not approach the positive values predicted if chromatin were uniformly supercoiled in the manner suggested by X-ray studies. The decay of the dichroism of chromatin, after termination of the electric pulse, is similar to that of DNA, indicating that chromatin retains a high degree of chain flexibility. These data, as well as previous flow dichroism studies (Ohba, 1966; Smart &; Bonner, 1971), suggest that chromatin is not predominantly supercoiled in solutions of low ionic strength. Evidence for a structurally heterogeneous model for chromatin is discussed.     

The absence of histone H1 from the chromatin fraction obtained by sonication of calf thymus nuclei under "quasiphysiological" ionic conditions.

The minor chromatin fraction was isolated from the sonicated calf thymus nuclei on the basis of its differential solubility in the "quasiphysiological" salt medium (0.1 M KCl-0.05 M NaCl-l mM MgCl2-1 mM CaCl2). Histone Hl is almost completely absent from this fraction. DNA isolated from this fraction occurs in three discrete low mol. wt. fragments. The fraction of chromatin which lacks histone Hl can also be obtained by two other methods. On of them consists in salt precipitation of the chromatin gel and its subsequent sonication. The second method includes precipitation of the sonicated chromatin gel by salts. In the first case the properties of the chromatin fraction which remains in the supernatant after centrifugation closely resemble those of the original salt-soluble nuclear fraction. The second method yields supernatant fraction also lacking histone Hl but containing heterogeneous DNA. Comparisons were also made of the sonically-solubilized nuclear fractions obtained in the complete salt medium and its mono and divalent cationic constituents.     

Synthesis of compositionally unique DNA by terminal deoxynucleotidyl transferase

Studies on the composition and characterization of DNA product(s) synthesized by calf thymus terminal deoxynucleotidyl transferase were performed using homopolymeric single-stranded, calf thymus double-stranded, and native DNA resident in calf thymus chromatin preparations as priming DNA species. Synthesis was carried out using equimolar concentrations of all four deoxynucleoside triphosphates as substrates and Mg²⁺ or Mn²⁺ as an effective divalent cation. Irrespective of the nature of the priming DNA or the divalent cation, the DNA product contained 60–70% dGMP residues, 10–15% each of the two pyrimidine residues, and 5–10% dAMP residues. The product synthesized using chromatin DNA as initiator was predominantly single-stranded and its synthesis was resistant to actinomycin D. The predilection of terminal deoxynucleotidyl transfease to synthesize dGMP-rich products on natural or homopolymeric DNA primers suggests that such products may represent biologically important recognition signal sequences.     

Modulation of chromatin superstructure induced by poly(ADP-ribose) synthesis and degradation

It has been demonstrated recently by Poirier et al. (Poirier, G. G., de Murcia, G., Jongstra-Bilen, J., Niedergang, C., and Mandel, P. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 3423-3427) that poly(ADP-ribosyl)ation of pancreatic nucleosomes causes relaxation of the chromatin superstructure through H1 modification. The in vitro effect of poly(ADP-ribose) synthesis and degradation on calf thymus chromatin was investigated by the time course incorporation of ADP-ribose, electron microscopy, analytical ultracentrifugation, and autoradiography of the protein acceptors. Purified calf thymus poly(ADP-ribose) polymerase and partially purified bull testis poly(ADP-ribose) glycohydrolase were used. Degradation of ADP-ribose units on hyper(ADP-ribosyl)ated H1 by poly(ADP-ribose) glycohydrolase restores the native condensed chromatin superstructure. This reversible conformational change induced by poly(ADP-ribosyl)ation on nucleosomal arrangement could be one of the mechanisms by which the accessibility of DNA polymerases and/or excision-repair enzymes is favored, the native structure being fully restorable.     

Protein-protein and protein-DNA interactions in calf thymus nuclear matrix using cross-linking by ultraviolet irradiation

Nuclear matrices from calf thymus contained 30-50 protein species with one prominent band at 70 kilodaltons tentatively identified by its isoelectric point, apparent molecular weight, charge modification, and abundance as bovine lamin. The amount of DNA present in the matrix fraction was strongly dependent on the extent of digestion of the nuclei by micrococcal nuclease. The size of the DNA was higher than two kilobase pairs, although the chromatin DNA had been digested down to short oligonucleosomes. The lamin band was preferentially dissociated from isolated matrices during repeated treatment by 2 M NaCl or 5 M urea. Irradiation of calf thymus nuclear matrices at 313 nm induced protein-protein and protein-DNA cross-linking, as well as double-strand breaking of DNA, presumably at unprotected, protein-free regions. Lamin protein was more dramatically affected than other protein species by ultraviolet (UV) irradiation. In situ DNA hydrolysis, after the separation of the cross-linked matrix components on polyacrylamide-sodium dodecyl sulfate gels, followed by two-dimensional electrophoresis, showed lamin to be the major protein that was cross-linked to the DNA. Lamin molecules were also cross-linked by UV light to each other to form lamin homo-oligomers. A discrete size DNA fragment of approximately 450 base pairs is protected by lamin homo-oligomers from breakdown during UV irradiation. It is proposed that the direct contact between lamin and DNA found in this study is responsible for anchoring chromatin loops (domains) to a stable, immobile matrix structure.     

Accessibility of the minor groove of DNA in chromatin to the binding of antibiotics netropsin and distamycin A

The interaction of the antibiotics distamycin A, distamycin analogue and netropsin with chromatin of calf thymus has been studied by circular dichroism measurements and by gel filtration. The minor groove of DNA in chromatin is accessible by 83–89% to the binding of these antibiotics as compared with that of free DNA. The present results combined with our data on the methylation of chromatin with dimethylsulphate [3] strongly suggest that the minor groove of DNA in chromatin is not occupied by chromatin proteins.Abbreviations DM distamycin A - DM₂ analogue of distamycin - Nt netropsin - CD spectra circular dichroism spectra     

Modification of histone binding in calf thymus chromatin by protamine.

When calf thymus chromatin is incubated with protamine, the protein binds to DNA, forming a chromatin-protamine complex. The binding reaches a saturating level at the weight ratio of protamine to DNA of approximately 0.5. Although the saturated binding of protamine to DNA does not cause major displacement of histones from calf thymus chromatin, examination of the dissociation profiles by salt in combination with urea of protamine-treated chromatin shows that the histone-DNA interactions are markedly altered by such binding. The dissociation of histones from the chromatin-protamine complex requires less NaCl but the same concentration of urea as that for untreated chromatin, suggesting that the electorstatic interactions between the histones and DNA are decreased as a result of protamine binding. When protamine concentration is increased beyond that required for saturated binding to DNA during in vitro exposure of calf thymus chromatin to protamine, lysine-rich histone is completely displaced.     

Suppression of nucleic acid synthesis in chromatin of Spirogyra during conjugation process

Marked changes in RNA- and DNA-synthesizing activities of thechromatin fraction were found to be connected with the conjugationprocess of Spirogyra sp. RNA-synthesizing activity of the chromatin fraction was muchlower in conjugating and zygote cells than in vegetatively growingcells. No significant differences were observed in the templateactivities of purified DNA prepared from vegetatively growing,conjugating, and zygote cells. The activities of DNA purifiedfrom vegetatively growing and zygote cells and calf thymus werestrongly inhibited at different levels by binding with histonesprepared from vegetatively growing and zygote cells. DNA-synthesizingactivity of the chromatin fraction of conjugating or zygotecells was much lower than that of vegetatively growing cells. (Received December 24, 1971; )     

Histones from Trypanosoma lewisi nuclei]

A preparation of total histones has been isolated for the first time from the purified fractions of T. lewisi cell nuclei and characterized in terms of its chemical composition and RNA-polymerase activity. A special attention during the isolation procedure was given to the repression of proteolytic degradation of the histones. The amount of protein in the chromatin is equivalent to that of DNA. The amino acid composition and heterogeneity of the protein during polyacrylamide gel electrophoresis in an acid system and in the presence of sodium dodecyl sulfate are typical for histones. Using two-dimensional electrophoresis, differential staining of electrophoregrams and ion-exchange chromatography on CM-cellulose the total preparation has been found to be made up of five fractions: two -- arginine-rich (one of them identical to histone H4, the other being similar to histone H3 from calf thymus); two -- moderately lysine-rich fractions, slightly differing in their properties from histones H2A and H2B from calf thymus, and one specific fraction with mol. weight of 16 000 and an extremely high positive charge. The above methods in combination with specific extraction have been used to demonstrate the absence of a typical lysine histone in the preparation, which is correlated with the absence of typical methaphase chromosomes during mitosis in T. lewisi.