Effect of formaldehyde on the circular dichroism of chicken erythrocyte chromatin.

Formaldehyde (HCHO) fixation of chicken erythrocyte chromatin produces a marked decrease in its positive circular dichroism (CD), above 260 nm, and the appearance of s small negative ellipticity around 295 nm. The ultraviolet spectrum of chromatin is unaffected, nor does HCHO produce any changes in the uv or CD spectra of chicken erythrocyte DNA. The extent of the circular dichroism transition from the native chromatin to the suppressed spectrum is dependent on the concentration of HCHO and salt concentration. The kinetics of the reactions are complex, implicating at least two reactive species. Studies of the reaction of HCHO with chromatin in ethylene glycol and CD measurements of aqueous chromatin solution with added glutaraldehyde preclude simple dehydration and general cross-linking effects as causes of the CD changes observed. The results are interpreted as indicating a conformational change of the DNA in chromatin caused by histone-DNA or histone-histone cross-linking.     

The conformation of proteins in chromatin. A circular dichroism study below 250 nm.

This paper is an investigation of the circular dichroism (CD) spectra of DNA and protein in chromatin. The circular dichroism (CD) of chromatin below 250 nm is due to DNA and protein peptide chromophores. The spectrum in this region is resolved into contributions from salt-extractable proteins (histone and non-histone proteins extractable with sodium chloride), residual non-histone proteins (not extractable with 3 M sodium chloride), and DNA. Below 250 nm, DNA in chromatin has the same CD spectrum as DNA free in solution, in contrast to the CD of DNA above 250 nm (Hjelm, R. and Huang, R. C., (1974), Biochemistry 13, 5275). Histones and salt-extractable non-histone proteins in chromatin are seen to have an average CD like those observed for globular proteins. The average CD of the residual non-histone proteins is consistent with a population of proteins with more extended conformation. The CD of each of these components is found to be the same in chromatins isolated from tissues having different nuclear synthetic activities: chick embryo brain, pig cerebellum, myeloma K41, calf thymus, and chicken erythrocyte.     

Phosphorylated protamines. II. Circular dichroism of complexes with DNA, dependency on ionic strength.

The influence of protamine phosphorylation upon the conformation of nucleoprotamine complexes was studied at different ionic strengths using circular dichroism. The sharp onset of CD spectral changes upon decreasing the NaC1 concentrationwas correlated with the beginning of complex formation and can be used to determine apparent binding affinities in terms of a critical ionic strength. It is show that phosphorylation strongly reduces the binding strength of protamines towards DNA. Directly mixed and reconstituted complexes reveal differences in their CD spectra, which decrease with increasing ionic strength. Spectra of complexes between threefold phosphorylated clupeine Z and DNA obtained by reconstitution or direct mixing at higher ionic strength resemble the phi-type spectra of DNA and are unique for the phosphorylated species. The implications of protamine phosphorylation for chromatin or DNA condensation havebeen discussed.     

A contribution of nonhistone proteins to the conformation of chromatin.

1. Changes in circular dichroism (CD) spectra and thermal melting profiles of guinea pigliver DNA reassociated with histones and/or nonhistone proteins from the cerebral of liver chromatin are described. 2. In the DNA-histone complex, positive ellipiticity in the CD spectrum at 260-300 nm is progressively lod by a red-shift of the crossover point at around 260 nm. DNA in this complex is thermally stabilised to a considerable extent, but not to such a full extent as is shown with DNA in native chromatin. 3. DNA-nonhistone complex in 0.14 M NaCl is, in contrast to DNA-histone complex, not precipitable by centrifugation at 20 000 X g. DNA in this complex shows only a slight reduction in ellipticity at 260-300 nm, and a very weak thermal stabilisation. 4. Characteristics in the CD spectrum of the native chromatin are most satisfactorily reproduced in the DNA-histone-nonhistone complex. These include a large decrease in ellipticity at 260-300 nm, a red-shift of the crossover point at around 260 nm, and a slight negative band at around 305 nm. Also, DNA in this complex is thermally stabilised to the extent comparable with DNA in the native chromatin. 5. Addition of nonhistone proteins to the preformed DNA-histone complex in 3 M urea renders a half of the complex, named DNA-histone(-nonhistone), unprecipitable upon centrifugation at 20 000 X g in 0.14 M NaCl. CD spectrum and thermal melting profile of the precipitable DNA-histone(-nonhistone) complex are similar to those of the DNA-histone-nonhistone complex, while in the unprecipitable DNA-histone(-nonhistone) comples, the ellipticity at 260-300 nm is significantly elevated and the highest melting transition (at 80 degrees C) is lacking. 6. The CD spectrum of native cerebral chromatin closely resembles that of unprecipitable DNA-histone(-nonhistone) complex, while in liver chromatin, the spec.trum is an intermediate between those of the unprecipitable and pn of chromatin by nonhistone proteins. Cerebral nonhistone proteins bind to DNA and to the DNA-histone complex more extensively than liver nonhistone proteins. 7. It is concluded that, although the basic conformation of DNA in native chromatin is determined largely by histones, nonhistone proteins also play an individual role. There is also an indication that nonhistone proteins exert an organ-specific modification of chromatin superstructure.     

Complexes of DNA with histones f2a2 and f3. Circular dichroism studies.

Two histones from calf thymus, the slightly lysine-rich histone f2a2 and the arginine-rich f3, were combined separately, with homologous DNA. The complexes were reconstituted by means of guanidine hydrochloride gradient dialysis, and their circular dichroic (CD) spectra were examined in 0.14 M NaCl. The CD spectra of f2a2-DNA complexes are characterized by a positive band at 272 nm which is blue-shifted and greatly enhanced relative to the corresponding band for native DNA. This type of CD change was noted previously with f2a1-DNA and f2b-DNA complexes. In contrast, f3 histone causes only minor distortions in the DNA CD spectrum, and their character depends upon the state of the two sulfhydryl groups in f3. When the cysteines are reduced, f3-DNA complexes have a slightly increased positive band with a small blue shift; when oxidized disulfide is the predominant form, this CD band becomes slightly smaller than native DNA value. This laboratory has now examined complexes reconstituted from DNA and all five histones of calf thymus. The sum of the CD spectra of these complexes, although very similar to the CD curve for reconstituted complexes containing whole histone, does not approximate that of chromatin; the consequence of this observation is discussed.     

Effects of shearing on chromatin structure

The effects of mechanical shearing on chromatin structure were investigated by using thermal denaturation and circular dichroism (CD) spectroscopy. Under ordinary conditions of mechanical shearing used for preparation of soluble chromatin, we observed only minor changes (less than 10%) of chromatin properties with respect to (a) absorption melting curves, (b) CD spectra, (c) CD melting curves and (d) histone transfer from chromatin to exogenous DNA. Such small pertubation of structural properties could be due to the generation of free ends when a large chromatin was cut into smaller fragments and by weakening the binding of histones to DNA near these free ends. In addition to mechanical shearing, sonication was used to shear some samples of chromatin. The effect of sonication on chromatin structure was investigated by the same physical methods used for mechanically sheared chromatin. The results indicate that sonication only slightly changes the chromatin properties with respect to CD spectra, similar to the results obtained by mechanical shearing, but sonication at high settings has a greater effect on the thermal denaturation property of chromatin as contrasted to our results from mechanically sheared chromatin.     

Changes in chromatin structure induced by EDTA treatment and partial removal of histone H1.

Electron microscopy shows that EDTA treatment or partial removal of histone HI converts 200-250 A chromatin fibres characteristic for native chromatin, isolated in low ionic strength conditions into fibres consisting of nucleosomes connected by segments of DNA. This structural transition is accompanied by an increase in the amplitude of positive band of CD spectra at 280 nm. Comparison of electron microscopic, thermal denaturation and electrophoretic data suggests that multiphasic character of melting curves, observed for chromatin, lacking histone HI is due to the removal of histone HI and destabilisation of the DNA segments, connecting nucleosomes. It is also shown that bivalent cations play an important part both in the stabilisation of 200 A globules and of nucleosomes.     

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     

Chromatin models. The ionic strength dependence of model histone-DNA interactions: circular dichroism studies of lysine-leucine polypeptide-DNA complexes.

The ionic strength dependence of the complexes between DNA and both random, (Lysx, Leuy)n, and block copolymers, (Lysx)n(Leuy)m, of lysine and leucine, with different amino acid compositions, was studied using circular dichroism (CD) as the probe to detect conformational differences in these complexes relative to native DNA. It was found that the CD spectra of complexes of both the random (Lys84, Leu16)n and block (Lys85)n(Leu15)m copolymers with DNA show a very sharp ionic strength dependence. The maximum altered CD spectrum for the complexes with the block copolymer was found to occur at the same ionic strength as that for poly(L-lysine)-DNA complexes, while the maximum CD change for the random copolymer complex occurred at a slightly lower ionic strength. This sharp dependence of the CD change on the ionic strength was found to be independent of the polymer/DNA ratio, r, for each individual copolymer. The CD spectra for these complexes at optimum NaCl concentration resemble those of the psi spectra of DNA [Jordan, C. F., Lerman, L.S., and Venable, J.H. (1972), Nature (London), New Biol. 236, 67]. The complexes of the random copolymer, (Lys68, Leu32)n, with DNA (r=0.25) at 0.15 M NaCl and below have CD spectra that resemble the A-form DNA spectra. The ionic strength dependence of the CD spectra of this complex is not as sharp as observed with the above polymers and has a broad positive plateau. It is suggested that both the CD spectra of these complexes reflect the phenomena of DNA condensation into a higher order asymmetric structure (folded and compact). The block copolymer, (Lys77)n(Leu23)m, complexes with DNA show very slight alterations in the CD spectra, with respect to native DNA. It appears that the long Leu sequence at one end of such copolymers may be unpropitious for causing the polypeptide-DNA complex to condense into a higher order asymmetric structure. Thus the importance of the distribution of hydrophobic residues, in the copolypeptides of Lys, is shown for causing condensation of complexes with DNA. The relevance of these findings to histone-DNA complexes in chromatin is discussed.     

Circular dichroism spectra and ethidium bromide binding of 5-deoxybromouridine-substituted chromatin.

Changes in CD spectra of 5-deoxybromouridine (brUdRib)-substituted chromatin depend on the extent of thymidine replacement by brUdRib. With 20 and 14 per cent replacement, a blue shift and a marked increase in positive ellipticity are reported in the CD spectra of brUdRib-substituted chromatin, while with 4.9 per cent replacement little effect is noticed. BrUdRib-substitution does not affect the number of ethidium bromide primary binding sites of chromatin.     

Circular dichroism of micrococcal nuclease-treated calf thymus chromatin (soluble chromatin) in presence of CH3HgOH

Exposing (soluble) calf thymus chromatin and, as reference, protein-free native calf thymus DNA (both in 0.01 M Na+, pH 6.8, 25 degrees C) to increasing concentrations of CH3HgOh produces cooperative transitions in their CD spectra. In the case of chromatin, and there especially at low concentrations of methylmercury, they are due to reactions affecting the relative orientation of the bases in the constituent DNA, without disrupting base-pairing. In the case of protein-free DNA, and with chromatin at higher methylmercury concentrations, the CD changes signal collapse of the DNA secondary structure. Primary data (molar ellipticities [theta], zero-ellipticity points, and rotational strengths R) are presented as a function of methylmercury concentration and wavelength. The results are discussed in relation to previous findings of this laboratory regarding methylmercury-DNA and methylmercury-chromatin interactions, and it is pointed out that the structural alterations observed with chromatin at low levels of methylmercury may very well be the primary events in a chain that is responsible for the teratogenic and clastogenic damages caused by organic mercury.     

Quasielastic light scattering by biopolymers. IV. Tertiary collapse of calf thymus DNA in 5.5M LiCl

Circular dichroism has been commonly employed to infer the conformation of DNA in solution. The basis of the conformational assignments is the work of Tunis-Schneider and Maestre, wherein CD spectra of DNA were obtained under conditions comparable to those employed in the x-ray diffraction studies of A-, B-, and C-DNA. It has recently been suggested that the CD spectrum of DNA in chromatin, which is similar to the CD spectrum of the C-form DNA, is a superposition of the normal B-DNA spectrum and a single negative band, centered at 275 nm. This negative band is qualitatively identical to the spectrum for condensed Ψ-form DNA. We have employed the hydrodynamic methods of quasielastic light scattering and sedimentation velocity to determine the extent of DNA tertiary structural alteration in 5.5M LiCl as a possible explanation of the C-form CD spectrum. These studies suggest an eightfold contraction of the Stokes hydrodynamic volume for calf thymus DNA in going from 0.4M NH₄Ac to 5.5M LiCl, with no change in molecular weight. The estimated maximum presistence length of DNA in 5.5M LiCl is estimated to be 20.0 nm compared to the “minimum” value of 44.7 nm in NaCl solutions. The value 20.0 nm corresponds to a maximum radius of 16.7 nm for a “continuously coiled” cylinder of DNA, which compares with the value 5.0 nm of DNA in the nucleosome unit of chromatin.     

Conformation and properties of DNA–(Lys33, Leu67)100–(Orn)20 complex: A nucleohistone model

The synthesis and characterization of the block copolypeptide (Leu⁶⁷, Lys³³)₁₀₀Orn₂₀, a synthetic model of histone, are reported. In neutral aqueous solutions, 80% of the etheropolypeptide block assumes an α-helical conformation, whereas the polyornithine block is in a random-coil conformation. In the association complexes with DNA, melting and titration experiments, as well as CD results, indicate that the polyornithine block interacts with DNA, whereas at least 2/3 of the lysine residues of the (Leu, Lys) moiety are excluded from the direct binding with DNA. CD spectra of the association complexes reveal significant differences from those obtained with DNA–polyornithine and DNA–polylysine complexes but substantial similarities with CD spectra of native and reconstituted nucleohistones. In contrast to DNA–polyornithine complexes, the CD spectra of the ternary complexes, copolypeptide–DNA–ethidium bromide, indicate a strong reduction of the dye intercalation. The low-angle x-ray diffraction pattern, reminiscent of that of chromatin, reveals the presence of a superstructure in these complexes. The results obtained are discussed in connection with the expected structural features of the model.     

Ionic strength-dependent conformational transitions of chromatin. Circular dichroism and thermal denaturation studies

High-molecular-weight chicken erythrocyte chromatin was prepared by mild digestion of nuclei with micrococcal nuclease. Samples of chromatin containing both core (H3, H4, H2A, H2B) and lysine-rich (H1, H5) histone proteins (whole chromatin) or only core histone proteins (core chromatin) were examined by CD and thermal denaturation as a function of ionic strength between 0.75 and 7.0 × 10^?3M Na⁺. CD studies at 21°C revealed a conformational transition over this range of ionic strengths in core chromatin, which indicated a partial unfolding of a segment of the core particle DNA at the lowest ionic strength studied. This transition is prevented by the presence of the lysine-rich histones in whole chromatin. Thermal-denaturation profiles of both whole and core chromatins, recorded by hyperchromicity at 260 nm, reproducibly and systematically varied with the ionic strength of the medium. Both materials displayed three resolvable thermal transitions, which represented the total DNA hyperchromicity on denaturation. The fractions of the total DNA which melted in each of these transitions were extremely sensitive to ionic strength. These effects are considered to result from intra- and/or internucleosomal electrostatic repulsions in chromatin studied at very low ionic strengths. Comparison of the whole and core chromatin melting profiles indicated substantial stabilization of the core-particle DNA by binding sites between the H1/H5 histones and the 140-base-pair core particle.     

A circular dichroism study of DNA-basic peptides associations in the absence or in the presence of Ca++.

The interaction of DNA with basic peptides (Lys methyl ester*, Lys2, (Lys)2methyl ester) has been studied by circular dichroism. The changes of the DNA CD spectra in the presence of peptides are interpreted as a transconformation from the B form to the C form of DNA. The presence of Ca++ in the mixture induces a supplementary transconformation. These observations suggest Ca++-basic peptides-DNA complexes as a structural model for chromatin.     

Circular dichroism of nucleic acid monomers. I. Calculated adenosine and 2′-deoxyadenosine CD spectra

A combination of the DeVoe and Kirkwood polarizability concepts is developed to calculate CD spectra of nucleic acid monomers. The method is perfectly general and applies to any system where the constituents have absorption properties which are widely separated in terms of frequency. The theory is applied to calculate the CD spectra of adenosine and 2′-deoxyadenosine conformers. Bond polarizabilities are evaluated for the ribosyl moiety of adenosine, as a function of glycosidic rotational angles and polarizability anisotropies. It is found that a wide range of C-C and C-O bond polarizabilities give similar CD results. Isotropic atom polarizabilities are also evaluated. It is found that the CD results using these polarizabilities do not differ significantly from those obtained with bond polarizabilities. The CD spectra of adenosine and 2′-deoxyadenosine are calculated for three x-ray diffraction determined geometries: A-form RNA, B-form DNA, and C-form DNA. The results indicate that the monomer CD spectra are strongly dependent on the precise geometry and appear to be of importance in understanding the spectra of oligomers and polymers. The deoxyadenosine conformers are found to have calculated CD spectra which are less intense than those of the ribosyl conformers. These results indicate that the measured differences between the CD magnitudes of ribo- and deoxyriboadenosine are due to the presence or absence of the 2′-hydroxyl. Weighted averaged adenosine CD spectra are calculated with the aid of probability distributions from conformational energy calculations. The results suggest a new method for obtaining empirical monomer parameters for use in optical calculations. The calculations in this paper indicate for the first time that DeVoe theory, in combination with the Kirkwood theory, provides a useful method for the calculation of the CD spectra of nonpolymeric molecules.     

Binding of progesterone by rat uterus in vitro

Circular dichroism (CD) spectra have been determined for chromatin fractions obtained by ECTHAM-cellulose chromatography. The molecular ellipticity at the positive long wavelength maximum is about 3000 deg cm²/dmol for early-eluted chromatin fractions, thought to be relatively repressed $\text{in vivo}$, and 5000–6000 deg cm²/dmol for late-eluted chromatin fractions, those thought to be preferentially transcribable $\text{in vivo}$. CD bands in the peptide bond spectral region also differ for the two chromatin fractions, early-eluted chromatin having a more helical conformation for proteins. In addition to previously known differences in protein content, the biological activity of a native chromatin fraction can now be correlated with the conformation of its DNA.     

Superstructure and CD spectrum as probes of chromatin integrity.

Two types of chromatin were extracted from the same stock of rat liver nuclei by a short exposure to micrococcal nuclease and by shearing respectively. These two materials which are identical in their protein/DNA content and by the presence of the five histones, were compared by means of circular dichroism and electron microscopy. Under the electron microscope and in absence of any divalent cation a superstructure of the unfixed chromatin fiber can be viewed only with native material but is no more present in sheared one. The increase of CD signal at 280 nm (from 2000 to about 4000 cm2 deg.dmole-1) in the case of sheared chromatin is not related to the loss of superstructure but to the structural changes of DNA inside the nucleosomal core which are always produced by shearing. These two correlated observations offer new sensitive probes of the integrity of any native or reconstituted chromatin.     

Spectral analysis of chromatin and its components in the vacuum ultraviolet region of the spectrum

Electronic absorption spectra of thin films of chromatin and chromatin components in ultraviolet (140-280 nm) were investigated. The absorption coefficients mu (lambda) of chromatin, nucleosomes with and without histone H1, total histones (TH), DNA were compared. The spectra of nucleosomes and chromatin differ from summary spectra of DNA + TH. The lack of additivity of absorption coefficients at different wavelengths may be explained by different conformational changes of free DNA, TH and DNA, TH in nucleosomes and chromatin during the process of drying aqueous solutions for the preparations of thin films. The obtained mu (lambda) values are necessary for the estimation of the DNA and TH parts of absorption in chromatin and nucleosomes in the investigations of UV and VUV irradiation damages.