Interaction of histone f2al fragments with deoxyribonucleic acid. Circular dichroism and thermal denaturation studies.

The glycine-arginine-rich histone, f2al (IV) (102 amino acids), from calf thymus was cleaved at residue 84 with cyanogen bromide. Complexes containing homologous DNA and each f2al fragment were reconstituted by means of Gdn-HC1 gradient dialysis. The circular dichroic (CD) spectra of these complexes were all examined in 0.14 M NaC1. The CD spectra of the DNA-f2al fragment complexes did not differ appreciably from that of DNA alone in the wavelength region above 240 nm. However, intact f2al-DNA complexes yield CD spectra which differ significantly (enhanced, blue-shifted, 273-nm band) from that of native DNA (Shih and Fasman, 1971). The small C-terminal fragment (85-102) was bound weakly to DNA under the conditions used. However, the large basic N-terminal fragment (1-83) was bound as well to DNA as was whole f2al, but produced no CD distortion. The conformation of the N-terminal fragment, unlike intact f2al, was not changed upon increasing the ionic strength to 0.14 M NaF. These results complement previous studies on f2al and its N-terminal CNBr fragment (Ziccardi and Schumaker, 1973).Thermal denaturation of the complexes in 2.5 X 10(-4) M EDTA was monitored simultaneously by changes in the absorption and CD spectra. All complexes showed a thermal transition at 45 degrees (Tml), attributable to the melting of free, double-stranded DNA. In addition, f2al-DNA and N fragment-DNA complexes displayed melting phenomena at 88 and 78 degrees (Tm2), respectively, caused by the denaturation of the histone-bound DNA. This difference in Tm2 constitutes further evidence that loss of the 18-amino-acid carboxyl end segment of f2al prohibits the unique type of interaction which occurs between DNA and the intact histone.     

Nucleosome core particles of calf thymus, Tetrahymena, and the reconstituted hybrid. Their structure reflects the nature of the histone octamer

The circular dichroism spectra and the thermal denaturation profiles of the nucleosome core particles isolated by micrococcal nuclease digestion from nuclei of calf thymus and the protozoan Tetrahymena pyriformis were compared with those of the homogeneous and hybrid core particles reconstituted from calf core DNA and either calf or Tetrahymena histone octamer. The core DNA was obtained from the calf core particle, and both the histone octamers were reconstituted from the acid-extracted four core histones of calf thymus or Tetrahymena, whose amino acid sequences show the largest differences hitherto known. The reconstituted homogeneous core particle was identical in both the physical properties with the isolated calf core particle, showing that the correct reconstitution was achieved. The circular dichroism spectra of the calf and Tetrahymena core particles and the hybrid core particle showed no essential differences, indicating that the three core particles have the same overall structure. The derivative thermal-denaturation profiles, however, clearly differed; the calf core particle showed two melting transitions at 60 degrees C and 72 degrees C, while the Tetrahymena and hybrid core particles showed the same three transitions at 48-50 degrees C, 60-61 degrees C, and 72 degrees C. Thus, the thermal denaturation properties of nucleosome core particles do not reflect the nature of DNA, but rather that of the histone octamer bound to the DNA. We conclude that the Tetrahymena histones are more weakly bound to the DNA than the calf thymus histones in the same overall structure of nucleosomes.     

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.     

Interactions of Hg(II) ions with DNA as revealed by CD measurements.

The circular dichroism spectra of Hg(II) complexes with native calf thymus DNA, chemically methylated Streptomyces chrysomallus DNA and with Ag(I)-DNA complexes were measured in the region of 220 - 340 nm. As a main result a conversion of the conservative CD spectrum of DNA to a distinct nonconservative type of CD spectrum for the complexes occurs with increasing Hg(II) concentration. The CD spectra of the Hg(II) complexes as well as some additional arguments strongly support the idea, that DNA in the complex undergoes a structural transition to a more condensed state with 4 -like character.     

The role of histone H2B from sea urchin sperm in the association of reconstituted minichromosomes

A comparative study of the condensation of reconstituted complexes of circular SV40 DNA with core histones from calf thymus and sea urchin sperm was performed using sedimentation and electron microscopic techniques. It is shown that in low ionic strength solutions both types of complexes are similar to native minichromosomes. In the region from 0.08 to 0.16 M NaCl the complexes of SV40 DNA with thymus histones form small compact particles. By contrast, the compaction of the SV40 DNA complexes with sperm histones results in the formation of giant intermolecular associates. The results obtained may mean that histone H2B of sea urchin sperm participates in the formation of a higher order structure in sperm chromatin.     

DNA sequence directs placement of histone cores on restriction fragments during nucleosome formation.

Restriction fragments, 203 and 144 base pairs in length, bearing the Escherichia coli lac control region have been reconstituted with the core histones from calf thymus to form nucleosomes. By several criteria the reconstituted nucleosomes are similar to native nucleosomes obtained by micrococcal nuclease digestion of calf thymus nuclei. However, sensitive nuclease digestion studies reveal subtle and important differences between native monosomes and the lac reconstitutes. Each reconstitute consists mainly of nucleosomes containing histone cores placed nonrandomly with respect to the DNA sequence. The shorter reconstitute forms asymmetric nucleosomes as evidenced by the DNase I digestion pattern. Exonuclease III digestion followed by 5'-end analysis of the larger reconstitute suggests that, of the many possible arrangements of histone core with DNA sequence, only two are highly favored.     

Interactions between arginine-rich histones and deoxyribonucleic acids. I. Thermal denaturation.

Physical properties of histone-DNA complexes very often depend upon the method of complex formation. In an attempt to make the studies of histone-DNA interactions more relevant to biological systems, results from thermal denaturation of native chromatin were used as references for determining how closely a given histone-DNA complex approaches its native state in chromatin. In the case of arginine-rich histones H3 (III or f3) and H4 (IV or f2a1), four methods were used for making complexes with calf thymus DNA: (A) NaCl gradient dialysis with urea; (B) NaCl gradient dialysis without urea; (C) direct mixing in 2.5 x 10(-4) EDTA, pH 8.0; and (D) direct mixing in 0.01 M sodium phosphate, pH 7.0. It was observed that a complex made by direct mixing in phosphate (method D) is closer to the native than is one made by direct mixing in EDTA (method C) than the one made by gradient dialysis with urea (method A) or without urea (method B). Regardless of the method used for complex formation, no substantial differences were observed between complexes with histone H3 dimer with disulfide bond(s) and a reduced H3 without disulfide bond, implying that perhaps a dimer with or without disulfide bond is a natural fundamental subunit in our experimental conditions. When the method of direct mixing in EDTA is used, the melting properties of the complexes vary only slightly with any one of the following H3 histones: from calf thymus, H3 without disulfide bond, H3 dimer, and H3 oligomer with disulfide bonds, also, from duck erythrocyte, H3 monomer and dimer. The complexes formed between DNA and a mixture of H3 and H4 by method D have melting properties similar to those of native chromatin. Since an equimolar mixture of histone H3 and H4 in 0.01 M phosphate, pH 7.0, was shown to form a tetramer (D'Anna, J.A., and Isenberg, I. (1974), Biochem. Biophys. Res. Commun. 61, 343), our results suggest that, a tetramer of H3 and H4, likely to be (H3)2(H4)2, formed from one H3 dimer and one H4 dimer, can bind DNA in a manner similar to that in native chromatin.     

Closely spaced nucleosome cores in reconstituted histone.DNA complexes and histone-H1-depleted chromatin

It has been demonstrated by digestion studies with micrococcal nuclease that reconstitution of complexes from DNA and a mixture of the four small calf thymus histones H2A, H2B, H3, and H4 leads to subunits closely spaced in a 137 +/- 7-nucleotide-pair register. Subunits isolated from the reconstituted complex contain nearly equimolar amounts of the four histones and sediment at 11.6S. On DNase I digestion both the reconstituted complex and the separated subunits gave rise to series of single-stranded DNA fragments with a 10-nucleotide periodicity. This indicates that the reconstitution leads to subunits very similar to nucleosome cores. Nucleosome cores closely spaced in a 140-nucleotide-pair register were also obtained upon removal of histone H1 from chromatin by dissociation with 0.63 M NaCl and subsequent ultracentrifugation. In reconstitution experiments with all five histones (including histone H1) our procedure did not lead to tandemly arranged nucleosomes containing about 200 nucleotide pairs of DNA. In the presence of EDTA, DNase II cleaved calf thymus nuclei and chromatin at about 200-nucleotide-pair intervals whereas in the presence of Mg2+ cleavage at intervals of approximately half this size was observed. The change in the nature of the cleavage pattern, however, was no longer found after removal of histone H1 from chromatin. This indicates that H1 influences the accessibility of DNase II cleavage sites in chromatin. This finding is discussed with respect to the influence of histone H1 on chromatin superstructure.     

Isolation and physico-chemical properties of native histone complexes: dimer (H2-H2B), tetramer (H3-H4)2 and octamer (H3-H4-H2A-H2B)2

The paper is concerned with the isolation of the native histone complexes: dimer (H2A-H2B), tetramer (H3-H4)2 and octamer (H3-H4-H2A-H2B)2 from the calf thymus chromatin under soft conditions (hydroxyl apatite) fractionation with the subsequent gel filtration). Parameters of hydroxyl apatite saturation with chromatin are determined. The complexes obtained are free of DNA and nonhistone proteins. Absorption spectra parameters, quantum efficiencies and fluorescence spectra typical of the corresponding histone oligomers are established. Comparison of free tyrosine fluorescence spectra with histone tyrosyl ones revealed a long-wave shift in the latter.     

Comparison between histones FV and F2a2 of chicken erythrocyte. II. Interaction with homologous DNA.

The conformation and stability of artificial complexes between chicken erythrocyte DNA and homologous histones FV and F2a2 was studied by circular dichroism (CD) and thermal denaturation followed by both absorbance and CD measurements. The complexes are made after a stepwise potassium fluoride gradient dialysis without urea and studied at low ionic strength (10-minus 3 M). 1) No structural changes of the DNA can be detected up to r equals 0.2 with FV and r equals 0.6 for F2a2. With FV at higher values of r the CD spectrum is altered, indicating the organization of DNA and histones in some kind of aggregate. 2) The conformation of histone molecules inside the complexes is not related to the ionic strength of the medium but to an effective ionic environment close to 0.1 M. This ionic strength would also correspond to the melting temperature of histone-covered DNA. 3) From the analysis of the absorbance melting profile the length of DNA covered with an histone molecule can be estimated. A good agreement is found between the negative charge of this piece of DNA and the net positive charge of the histone. 4) Since the CD transition at 227 nm occurs before the second absorbance transition at 280 nm, the DNA is stabilized no longer by native histone but partially or fully denatured histones. The helical regions of the histone molecule are not involved in the binding process, which appears to be almost purely coulombian and most likely related to some structural fit between the pattern of negative charges in the DNA helix and that of positive charges along the peptide chain.     

Circular dichroism of native and reconstituted nucleohistones

Circular dichroism (CD) spectra of histone, DNA and native, partial and reconstituted nucleohistone complexes in 0.01 M Tris buffer in which nucleohistone complexes appear to be intact and in 2 M NaCl, in which complexes are mostly dissociated, have been compared. The CD spectra in the longwave region, where the protein contribution is negigible, reveal differences in the spectra for DNA in nucleohistone complexes as compared with that of free DNA, the difference being roughly proportional to the protein DNA ratio. An analysis of the shape of the first positive CD maximum suggests that we are probably dealing here with slight conformational changes on DNA. Minor differences between the behavior of native and reconstituted complexes may be due to the effect of different conformational states of the protein component.     

Structure and histone composition of chromatin in Physarum polycephalum]

In Physarum polycephalum several degrees of organisation of deoxyribonucleoprotein fibres were found. The complexes of histones and the DNA duplex seem to "be packed" at first into a 100 A fibre and then into a 200 A fibre of DNP. In Ph. polycephalum the electrophoretic mobilities of histone fractions 4 and 6 are comparable to that of fractions f3/f2b and f2a1 of calf thymus, resp. Histone fractions 3 and 5 move a bit faster than fractions f1 and f2a2, resp. Thus, the myxomycete P. polycephalum is similar to higher eukaryotes as concerns the ultrastructure of chromatin and electrophoretic properties of histones.     

Immunological and circular dichroism studies of maleylated f-1 (A) histone and complexes with DNA

Complexes of calf thymus f-1 (A) histone and homologous DNA were examined by circular dichroism. The maleylation of f-1 (A) produces a polypeptide with decreased ability to modify the circular dichroism spectrum of f-1 (A)-DNA complexes. By the introduction of two to three maleyl groups per f-1 (A) molecule, the alteration of the DNA CD spectrum is reduced by nearly half compared to that induced by the native nonmaleylated f-1 (A). Similarly maleylation reduces the serological reactivity of the histone, i.e., the reaction of the maleylated f-1 (A) with specific complement fixing f-1 (A) antibodies. On the other hand, moderate maleylation of f-1 (A) improves the cross-inhibition of the f-2b-anti-f-2b reaction by native f-1 (A) while extensively maleylated f-1 (A) is inert with respect to the same reaction. These results are interpreted in terms of possible conformational changes induced in f-1 (A) by maleylation, partially due to decreasing the histone net charge and perhaps as well as removal of specific site charges necessary for correct binding and interaction. Such an interpretation is consistent with the altered CD spectrum of maleylated f-1 (A) (i.e., a decreased and slightly red-shifted [θ]₁₉₈) and moreover explains why maleylation of two to three lysines per f-1 (A) molecule hinders simultaneously the very different DNA-histone and histone-complement fixing antibody interactions.     

Analysis of the binding of C-reactive protein to chromatin subunits

C-reactive protein (CRP) is an acute phase serum protein in man. The functional activities of CRP, like Ig, include complement activation and enhancement of phagocytosis. CRP binding to several substrates, including phosphocholine, individual denatured histones, and chromatin, has been demonstrated. We previously demonstrated that CRP binding to chromatin is dependent on the presence of histone H1, despite the fact that CRP binds to purified individual histones H2A and H2B, as well as to H1. In this report we examined the binding of CRP to native sub-nucleosomal chromatin fragments. CRP binding to the H2A-H2B dimer and (H3-H4)2 tetramer was demonstrated and these reactions were inhibited by phosphocholine. However, no binding to the subnucleosome complexes (H2A-H2B)-DNA and (H3-H4)2-DNA was seen. Similarly, CRP binding to H1 was eliminated when H1 was reconstituted with DNA. The reconstitution of H1-depleted chromatin with H1 restored CRP binding. CRP binding to nucleosome core particles, as previously demonstrated by others, was confirmed. Therefore, the interaction of CRP with individual core histones does not appear to be responsible for the binding of CRP to native chromatin. However, binding to core particles could be mediated by differentially exposed determinants on H2A and H2B.     

Circular dichroism of films of polynucleotides

The CD spectra of films of the lithium salt of E. coli and calf thymus DNA, and alternating d-AT : AT were measured as a function of relative humidity. Films of the ammonium acetate salt of DNA were also measured. The ammonium films yield the previously reported A-form CD spectra. A possible explanation for the small magnitude of the 260-nm band of the A-form film spectra compared to double-stranded RNA spectra is that the film DNA is in a different conformation than RNA within the A family of conformations. At relative humidities of 92% or lower, a negative nonconservative CD spectrum with negative minima near 270 and 210 nm is observed with the lithium films. The magnitude of the minima varies from film to film. In films of DNA the magnitude ranges from a delta epsilon of ?5 to ?35; d-AT : AT films show magnitudes to ?300. CD spectra of this type are designated Ψ spectra. Similar spectra have been reported from reconstituted complexes of DNA and polylysine or f-1 histone. If the origins of the film and protein–DNA complex spectra are similar, the complex spectra are not the result of specific secondary structural changes induced in the DNA by the protein fraction. Theoretical analysis suggests that Ψ spectra are not the result of changes in the secondary or tertiary structure of DNA. Instead, the previously proposed explanation based on liquid crystals is favored. The DNA could form asymmetric structures with long-range periodicity. It is likely that the observed CD spectra of f-1 complexes are artifacts of DNA aggregation. The possibility that some other previously published spectra of protein–DNA complexes also reflect artifacts is suggested.     

Studies on interaction between histone V (f2c) and deoxyribonucleic acids.

Histone V (2fc) from chick erythroctes was used in the study of its interaction with DNA from various sources. Complexes between this histone and DNA were formed using the procedure of continuous NaCl gradient dialysis in urea. Two physical methods, namely thermal denaturation and circular dichroism (CD), were used as analytical tools. Thermal denaturation of nucleohistone V with chick or calf thymus DNA shows three melting bands: band I at 45-50 degrees corresponds to free base pairs; band II at 75-79 degrees, and band III at 90-93 degrees correspond to histone-bound base pairs. In histone-bound regions, there are 1.5 amino acid residues/nucleotide in nucleohistone V. In contrast, a value between 2.9 and 3.3 was determined for nucleohistone I (fl) (H. J. Li (1973), Biopolymers 12, 287). Similar melting properties have been observed for histone V complexed with bacterial DNA from Micrococcus luteus. Histone V binding to DNA induces a slight transition from a B-type CD spectrum to a C-type spectrum. Trypsin treatment of nucleohistone V reduces melting band III much more effectively than band II. Such a treatment also restores DNA to B conformation in the free state. Reduction of the melting bands of nucleohistone V by polylysine binding follows the order of I greater than II greater than III, accompanied by the increase of a new band at 100 degrees. When two bacterial DNAs of varied A + T (adenine + thymine) content simultaneously compete for the binding of histone V, the more (A " T)-rich DNA is selectively favored. Under experimental conditions described here, Clostridium perfringens DNA with 69% A + T is bound by histone V in preference to chicken DNA with 56% A + T although the latter has natural sequences for histone V binding.