Structure of DNA complexes with chromosomal protein HMGB1 and histone H1 in the presence of manganese ions: 1. Circular dichroism spectroscopy

Mechanisms of interaction of DNA with nonhistone chromosomal protein HMGB1 and linker histone H1 have been studied by means of circular dichroism and absorption spectroscopy. Both proteins are located in the internucleosomal regions of chromatin. It is demonstrated that the properties of DNA-protein complexes depend on the protein content and cannot be considered as a mere summing up of the effects of individual protein components. Interaction of the HMGB1 and H1 proteins is shown with DNA to be cooperative rather than competitive. Lysine-rich histone H1 facilitates the binding of HMGB1 to DNA by screening the negatively charged groups of the sugar-phosphate backbone of DNA and dicarboxylic amino acid residues in the C-terminal domain of HMGB1. The observed joint action of HMGB1 and H1 stimulates DNA condensation with the formation of anisotropic DNA-protein complexes with typical ψ-type CD spectra. Structural organization of the complexes depends not only on DNA-protein interactions but also on interaction between the HMGB1 and H1 protein molecules bound to DNA. Manganese ions significantly modify the mode of interactions between components in the triple DNA-HMGB1-H1 complex. The binding of Mn²⁺ ions weakens DNA-protein interactions and strengthens protein-protein interactions, which promote DNA condensation and formation of large DNA-protein particles in solution.     

The structure of the complexes of DNA with chromosomal protein HMGB1 and histone H1 in the presence of manganese ions. I. Circular dicroism spectroscopy

The mechanisms of interaction of the non-histone chromosomal protein HMGB1 and linker histone H1 with DNA have been studied using circular dichroism and absorption spectroscopy. Both of the proteins are located in the inter-nucleosomal regions of chromatin. It was demonstrated that properties of the DNA-protein complexes depend on the protein content and can not be considered as a simple summing up of the effects of individual protein components. Interaction of HMGB1 and H1 proteins is shown to be co-operative rather than competitive. Lysine-rich histone H1 facilitates the binding of the HMGB1 with DNA by screening the negatively charged groups of the sugar-phosphate backbone of DNA and dicarboxylic amino-acid residues in the C-terminal domain of the HMGB1 protein. The observed joint action of the and H1 proteins stimulates DNA condensation with formation of the anisotropic DNA-protein complexes with typical psi-type CD spectra. Structural organization of the complexes depends not only on the DNA-protein interactions, but also on the interaction between HMGB1 and H1 protein molecules bound to DNA. Manganese ions significantly modify the character of interactions between the components in the triple DNA-HMGB1-H1 complex. Binding of Mn2+ ions causes the weakening of the DNA-protein interactions and strengthening the protein-protein interactions, which promote DNA condensation and formation of large DNA-protein particles in solution.     

The structure of the complexes of DNA with non-histone chromosomal protein HMGB1 in the presence of manganese ions

The complexes of DNA - HMGB1 protein - manganese ions have been studied using circular dichroism (CD) technique. It was shown that in such three-component system the interactions of both the protein and metal ions with DNA differ from those in two-component complexes. The manganese ions do not affect the CD spectrum of free HMGB1 protein. However, Mn2+ ions induce considerable changes in the CD spectrum of free DNA in the spectral range of 260-290 nm. The presence of Mn2+ ions prevents formation of the ordered supramolecular structures specific for the HMGB1-DNA complexes. The interaction of manganese ions with DNA has a marked influence on the local DNA structure changing the properties of protein-binding sites. This results in the serious decrease in cooperativity of the DNA-protein binding. Such changes in the mode of the DNA-protein interactions occur at concentrations as small as 0.01 mM Mn2+. Moreover, the changes in local DNA structure induced by manganese ions promote the appearance of new HMGB1 binding sites on the DNA double helix. At the same time interactions with HMGB1 protein induce alterations in the structure of the DNA double helix which increase with a growth of the protein/DNA ratio. These alterations make the DNA/protein complex especially sensitive to manganese ions. Under these conditions the Mn2+ ions strongly affect the DNA structure that reflects in abrupt changes of the CD spectra of DNA in the complex in the range of 260-290 nm. Thus, structural changes of the DNA double helix in the three-component DNA-HMGB1-Mn2+ complexes come as a result of the combined and interdependent interactions of DNA with Mn2+ ions and the molecules of HMGB1.     

Basic polypeptides as histone models: circular dichroism of complexes of model polypeptides with DNA.

Circular dichroism (CD) was used to study the complexes of DNA (in 0.15M NaCl) with two polypeptides considered as models of the histone molecules. CD spectra in the region of DNA absorption were studied with respect to the concentration used for annealing and to the molecular weight and composition of the DNA used. The properties of supernatants after centrifugation of aggregated complexes were examined. The effect of selectively bound antibiotics (actinomycin D and netropsin) on CD sprectra of complexes was investigated. The induced CD of proflavine molecules bound to DNA in the various complexes was also studied. It was concluded that changes in the CD spectra of DNA in complexes with the polypeptides are due to the formation of chiral superstructures, even if some conformational changes of DNA molecules themselves may also be decisive in some cases. The superstructure is affected by the composition of DNA, the role of (G + C) rich segments being particularly important.     

Condensation of DNA by the C-terminal domain of histone H1. A circular dichroism study

The condensation of DNA by the C-terminal domain of histone H1 has been studied by circular dichroism in physiological salt concentration (0.14 M NaF). As the intact H1 molecule, its C-terminal domain induces the so-called psi state of DNA that is characterized by a nonconservative circular dichroism spectrum which is currently attributed to ordered aggregation of the DNA molecules. On a molar basis, intact H1 and its C-terminal domain give spectra of similar intensity. Neither the globular domain of H1 nor an N-terminal fragment, that includes both the globular and N-terminal domains, has any effect on the conservative circular dichroism of DNA. From these results it is concluded that the condensation of DNA mediated by histone H1 is mainly due to its C-terminal domain. The effect of the salt concentration and the size of DNA molecules on the circular dichroism of the complexes are also examined.     

Structure of transfection-active histone H1/DNA complexes

Relationships between the structure of transfecting complexes of histone H1 and DNA and their transfection efficiency were studied. Transfection activity proved to be connected to complex aggregates. Low speed centrifugation of the complexes resulted in loss of the transfection activity. The complexes/aggregates were active with high efficiency in a broad range of weight input ratios r _i (0.1<r _i<30). Using atomic force microscopy (AFM), the complexes were imaged at negative, nearly electroneutral and positive charge conditions. Electroneutral complexes at r _i=1 showed a multitude of different complex forms. Fibrillar, network-like and branched structures were frequently present in one complex. Strongly positive charged complexes had a toroidal appearance. All these different forms contributed to the high transfection efficiency. Cellular uptake is supposed to be by phagocytosis.     

The effect of Ca2+ ions on DNA compaction in the complex with non-histone chromosomal protein HMGB1

The analysis of absorption and circular dichroism spectra in UV and IR regions showed that Ca2+ ions interact both with the phosphate groups of DNA and with the HMGB1 protein. Not only negatively charged C-terminal part of the protein molecule participates in interaction with metal ions but also its DNA-binding domains. The latter fact leads to the change of the mode of protein-DNA interaction. The presence of Ca2+ ions prevents formation of ordered supramolecular structures, specific for the HMGB1-DNA complexes, though promotes intermolecular aggregation. The structure of the complexes between DNA and the protein HMGB1 lacking C-terminal tail appears to be the most sensitive to the presence of Ca2+ ions. The data obtained allow to conclude that Ca2+ ions do not play a structural role in the HMGB1/DNA complexes and the presence of these ions is not necessary to DNA compaction in such systems.     

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.     

Vibrational circular dichroism studies of the A-to-B conformational transition in DNA.

The vibrational circular dichroism (VCD) spectra of several natural DNAs as well as tRNA, poly(dG-dC).poly(dG-dC), and poly(dA-dT).poly(dA-dT) are reported for the base deformation modes in the IR region from 1700 to 1550 cm-1 for the polymers in D2O as well as in high alcohol dehydrating conditions. Spectra of both the B- and A-forms were identified. The A-form DNA VCD, not previously reported, has characteristics that can be found in the VCD spectra of RNAs as would be expected from the similarity of their structures. The VCD is sequence-dependent. Under the dehydrating conditions studied, poly(dA-dT)poly(dA-dT),poly(dA).poly(dT), and a high-A-T fraction natural DNA had a different bandshape from the other DNAs, which was similar to that of poly(rA).poly(rU). Poly(dG-dC).poly-(dG-dC) did not form an A-form in high-alcohol conditions but instead had a VCD spectrum much like that of its high-salt-induced Z-form. Qualitative differences seen experimentally between A- and B-form DNA VCD were suggested by the differences in the coupled oscillator VCD calculated for the two forms.     

Non-histone chromosomal protein HMG1 modulates the histone H1-induced condensation of DNA

Circular dichroic spectra revealed that the previously known regular, asymmetric condensation of DNA by H1 histone was modulated by HMG1, a nonhistone chromosomal protein. Under approximately physiological salt and pH conditions (150 mM NaCl, pH 7), ellipticities at 270 nm were observed as follows: DNA, 9 X 10(3) degree, cm2/dmol nucleotide; DNA X H1 histone complex (1:0.4, w/w), -37 X 10(3) degree, cm2/dmol nucleotide, and DNA X H1 X HMG1 complex (1:0.4:0.4 w/w/w), -52 X 10(3) degree, cm2/dmol. HMG1 by itself did not distort the spectrum of DNA, showing that the effect of HMG1 on the DNA X H1 complex was not simply the summation of individual effects of HMG1 and H1 on the DNA spectrum. The effect of added HMG1 on the spectrum of the preformed DNA X H1 complex depended on the amount of HMG1 added and developed slowly (a day) as if a structure required annealing. The ternary complex, DNA X HMG1 X 1, seemed to represent a specific structure, since its formation depeNded on the reduced sulfhydryl state of HMG1; the disulfide form of HMG1, which was shown by circular dichroism to contain more random coil than did the reduced form, had no effect on the circular dichroic spectrum of the DNA X H1 complex.     

Binding of recA protein to Z-form DNA studied with circular and linear dichroism spectroscopy

Binding of RecA to poly(dG-m5dC) and poly(dG-dC) under B- and Z-form conditions was studied using circular dichroism (CD) and linear dichroism (LD). LD revealed a quantitative binding of RecA to Mg2+-induced Z-form poly(dG-m5dC) with a stoichiometry of 3.1 base pairs/RecA monomer, which is slightly larger than the 2.7 base pairs observed for the B-form. The LD spectra indicate a preferentially perpendicular orientation of DNA bases and a rather parallel orientation of the tryptophan residues relative to the fiber axis in both complexes. The association rate of RecA to Z-form DNA was found to be slower than to B-form. CD measurements showed that the polynucleotide conformation is retained upon RecA binding, and CD and LD confirm that RecA binds to both forms of DNA. The Mg2+-induced Z-form is shown to be retransformed into B-form, both in free and in RecA-complexed polynucleotides by addition of NaCl, whereas the B----Z transition cannot be induced by addition of Mg2+ when the polynucleotide is complexed with RecA. From this it is inferred that RecA does not stabilize the Z-conformation of the polynucleotide but that it can kinetically freeze the polynucleotide in its B-conformation. On all essential points, the same conclusions were also reached in a corresponding study of unmethylated poly(dG-dC) with the Z-form induced by Mn2+.     

Complexes of DNA with trivalent metal ions in presence of manganese ions

The interaction of DNA with Fe3+, Al3+, Co(NH3)6(3+) in a solution containing MnCl2 was studied. It was shown that there exists a competition for the binding sites between Mn2+ and Al3+, while the binding of Mn2+ to DNA does not depend on the presence of Fe3+ and Co(NH3)6(3+) in solution. We proposed that Fe3+ and Co(NH3)6(3+) ions prefer to bind to phosphates, and Al3+ ions are capable to bind to the nitrogen bases of DNA.     

Particular structural role of H1 in complexes with DNA and comparison with H2A- and H4-DNA complexes investigated by IR linear dichroism.

The particular role of H1 in the structure of histone–DNA associations is shown by means of ir linear dichroism. H1–, H2A–, and H4–DNA complexes are studied for different histone: DNA input ratios and various relative humidities (r.h.). The measurement of the dichroic ratios allows one to determine the secondary structure of DNA in the complexes. It is shown that the progressive addition of histone H2A or H4 to DNA inhibits the structural B → A transition and DNA remains in a B-type form at low r.h. It is found that the B → A transition is inhibited for 19 or 26 base pairs of DNA per molecule of H2A or H4. The stabilization of DNA in a B-conformation by H2A and H4 has been also observed by H2B and H3 but with a different efficiency. In contrast, histone H1, which does not belong to the core of the nucleosomes in chromatin, leaves the DNA in H1–DNA complexes free to adopt an A conformation at low r.h. for H1/DNA ratios below 0.6/1. Thus a major difference in the structural role between histone H1 and histones belonging to the nucleosomal core with respect to the conformational flexibility of DNA in the histone–DNA complexes is demonstrated.     

Complexes of histone F1 with DNA in 0.15M NaCl. Circular dichroism and structure of the complexes

Conditions were studied under which aggregated complexes of histone F1 with DNA display a circular dichroic spectrum of nonconservative type characterized by two intense negative maxima at 269 nm and 209 nm. It was found that the intensity of the longwave band depends directly on the content of guanine plus cytosine and inversely on the molecular weight of DNA. The same type of dependence was found for DNA's of eukaryotic and bacterial origin. It appears that the formation of the nonconservative spectrum which can be ascribed to a certain structural form of DNA is caused by orientation of the DNA molecules in the aggregated complex and by specific interactions of the bases of the guanine–cytosine pair. The possible mechanism of formation of nonconservative dichroic spectrum is discussed with respect to special properties of guanine.     

The histone H3/H4.N1 complex supplemented with histone H2A-H2B dimers and DNA topoisomerase I forms nucleosomes on circular DNA under physiological conditions

We have fractionated the whole cell extract of Xenopus oocytes (oocyte S-150) and isolated the endogenous components required for DNA supercoiling and nucleosome formation. Histone H2B and the three oocyte-specific H2A proteins were purified as free histones. Histones H3 and H4 were purified 100-fold in a complex with the acidic protein N1. In the presence of DNA topoisomerase I or II, histone H3/H4.N1 complexes supercoil DNA in a reaction that is inhibited by Mg2+, and this inhibition is relieved by NTPs. The supercoiling reaction induced by H3/H4.N1 complexes is enhanced by free histone H2A-H2B dimers, which by themselves do not supercoil DNA. Nuclease digestions and protein analyses indicate that H3/H4.N1 complexes form subnucleosomal particles containing histones H3 and H4. Nucleosomes containing 146-base pair DNA and the four histones are formed when histones H2A and H2B complement the reaction.     

Basic polypeptides as histone models. Effect of conformation, base composition and methylation of nucleic acids on the interaction with H1 and histone models and on the circular dichroism of complexes.

Interaction of histone H 1 and models simulating histone chains was followed by monitoring the melting curves of supernatants after the sedimentation of aggregated complexes. In a mixture of two DNAs the histones reacted selectively with (A+T)-rich and non-methylated DNA, respectively. H 1 and (Ala-Lys-Pro)n also interacted preferentially with DNA in a mixture with double stranded RNA whereas (Lys30,Ala70)n did not show any selectivity. (G+C)-rich DNA in complexes showed CD spectra the intensity of which decreased with increasing DNA methylation to values comparable with these of complexes of (A+T)-rich DNA. In complexed with double stranded RNA only the polymer (Lys30,Ala70) displayed CD pattern similar to spectra of complexes with DNA. It was concluded that formation and structure of complexes depend selectively on the DNA conformation and base composition.