Nonhistone proteins HMG1 and HMG2 unwind DNA double helix.

In a previous communication we have shown that both HMG1 and HMG2 nonhistone proteins change the DNA helical structure and the binding of HMG1 and HMG2 to DNA induces a net unwinding equivalent of DNA double helix (Javaherian, K., Liu, L. F. and Wang, J. C. (1978) Science, 199, 1345-1346). Employing melting absorption technique, we now show that in the presence of salt HMG1 and HMG2 destabilize DNA whereas in the absence of salt, they both stabilize DNA molecules. Consequently the folded structure of HMG must play an important role in melting DNA. Furthermore, by measuring topological winding number using competition unwinding experiments, we conclude that HMG1 has a higher affinity for a single-stranded DNA relative to double-stranded DNA. These results together suggest that HMG1 and HMG2 unwind DNA double helix by local denaturation of the DNA base pairs. The net unwinding angles have been measured to be 22 degrees and 26 degrees per molecule of HMG1 and HMG2 respectively.     

Mg2+-dependent unwinding of DNA by nonhistone chromosomal protein HMG(1 + 2) from pig thymus as determined by DNA melting temperature analysis

In the previous studies with endonucleases specific for single-stranded DNA, we have indicated that the nonhistone chromosomal protein HMG(1 + 2) prepared from pig thymus has an activity to unwind DNA partially at low protein-to-DNA weight ratios (Yoshida, M. & Shimura, K. (1984) J. Biochem. 95, 117-124). In the present work, we have pursued the unwinding reaction by HMG(1 + 2) by thermal melting temperature analysis of DNA, and by investigating the effect of Mg2+ on the reaction. The melting temperature of DNA in the presence of HMG(1 + 2) at low protein weight ratios decreased in 2 mM Tris-HCl, pH 7.8, whereas it increased at higher ratios. The depressions of melting temperature by HMG(1 + 2) at low ratios were not observed either in the system of 2 mM Tris-HCl, pH 7.8, containing EDTA or in the system containing samples treated in advance with EDTA. An addition of Mg2+ to the system reproduced the depression of melting temperature at low protein-to-DNA ratios as well as the increase at higher ratios. Analysis by Mg2+-equilibrated gel filtration revealed that HMG(1 + 2) is a Mg2+-binding protein. However, the depression of melting temperature at low protein-to-DNA ratios was not due to removal of Mg2+ from DNA by HMG(1 + 2). From these results, it is concluded that HMG(1 + 2) causes a partial DNA unwinding detectable by thermal melting temperature analysis of DNA, and that Mg2+ is necessary for the unwinding reaction.     

Unwinding of DNA by nonhistone protein HMG1 and HMG2

The enzyme kinetic studies with endonucleases specific for single-stranded DNA and the thermal denaturation analyses of DNA showed that a high mobility group (HMG) nonhistone protein fraction HMG (1 + 2), composed of HMG1 and HMG2, has an activity to unwind DNA partially at low protein-to-DNA weight ratio. Isolated HMG1 and HMG2 have the same activity. Divalent cations such as Mg++ or Ca++ were necessary for the unwinding reaction. A peptide containing high glutamic and aspartic (HGA) region, isolated from the tryptic digest of HMG (1 + 2), unwound DNA depending on the presence of Mg++ or Ca++, suggesting that the HMA region in HMG protein is the active site for the DNA unwinding reaction. Poly-L-glutamic acid, employed as a model peptide of the HGA region, showed the activity. Finally, mechanisms of the DNA unwinding reaction by the HMG protein and possible role of the divalent cations are discussed.     

Influence of Amino Acids Shiff Bases on Irradiated DNA Stability In Vivo

To reveal protective role of the new Mn(II) complexes with Nicotinyl-l-Tyrosinate and Nicotinyl-l-Tryptophanate Schiff Bases against ionizing radiation. The DNA of the rats liver was isolated on 7, 14, and 30 days after X-ray irradiation. The differences between the DNA of irradiated rats and rats pre-treated with Mn(II) complexes were studied using the melting, microcalorimetry, and electrophoresis methods. The melting parameters and the melting enthalpy of rats livers DNA were changed after the X-ray irradiation: melting temperature and melting enthalpy were decreased and melting interval was increased. These results can be explained by destruction of DNA molecules. It was shown that pre-treatment of rats with Mn(II) complexes approximates the melting parameters to norm. Agarose gel electrophoresis data confirmed the results of melting studies. The separate DNA fragments were revealed in DNA samples isolated from irradiated animals. The DNA isolated from animals pre-treated with the Mn(II) chelates had better electrophoretic characteristics, which correspond to healthy DNA. Pre-treatment of the irradiated rats with Mn(II)(Nicotinil-l-Tyrosinate) and Mn(II)(Nicotinil-l-Tryptophanate)₂ improves the DNA characteristics.     

Domain-domain interactions in high mobility group 1 protein (HMG1).

The high mobility group protein HMG1 is a conserved chromosomal protein with two homologous DNA-binding domains, A and B, and an acidic carboxy-terminal tail, C. The structure of isolated domains A and B has been previously determined by NMR, but the interactions of the different domains within the complete protein were unknown. By means of differential scanning calorimetry and circular dichroism we have investigated the thermal stability of HMG1, of the truncated protein A-B (HMG1 without the acidic tail C) and of the isolated domains A and B. In 3 mm sodium acetate buffer, pH 5, the thermal melting of domains A and B are identical (transition temperature tm = 43 degrees C and 41 degrees C, denaturation enthalpies DeltaH = 46 kcal.mol-1). The thermal melting of protein A-B presents two nearly identical transitions (tm = 40 degrees C and 41 degrees C, DeltaH = 44 kcal.mol-1 and 46 kcal.mol-1, respectively). We conclude that the two domains A and B within protein A-B behave as independent domains. The thermal melting of HMG1 is biphasic. The two transitions have a different value of tm (38 degrees C and 55 degrees C) and corresponding values of DeltaH around 40 kcal.mol-1. We conclude that within HMG1, the acidic tail C is interacting with one of the two domains A and B, however, the two domains A and B do not interact with each other. At 37 degrees C, one of the two domains A and B, within HMG1, is partly unfolded, whereas the other which interacts with the acidic tail C, is fully native. The interaction free energy of the acidic tail C is estimated to be in the range of 2.5 kcal.mol-1 based on simulations of the thermograms of HMG1 as a function of the interaction free energy.     

A DNA study of rat liver oligonucleosomes enriched by transcriptionally active genes during induction due to the administration of an amino acid mixture

A highly active fraction of rat liver oligonucleosome DNA has been isolated and studied by means of thermal denaturation after induction by amino acid mixture or hydrocortisone. A considerable redistribution of DNA content has been shown in sucrose gradient fractions during these forms of induction. The changes are revealed in melting temperature, differential melting profile of DNA, isolated from actively transcribed chromatine fractions. Analysis of melting profiles shows changes of GC content of oligonucleosome DNA, suggesting that there are differences in activation during two studied forms of induction.     

Melting of DNA from tumor-bearing rats treated with <Emphasis Type="Italic">meso</Emphasis>-Tetra(4-N-hydroxyethylpyridyl)porphyrin and its Ag,Zn, and Co derivatives

The comparative study on the secondary structure of DNA isolated from the liver tissue of normal rats, as well as from the tumor tissue of rats both treated and untreated with porphyrins was carried out. meso- Tetra(4-N-hydroxyethylpyridyl)porphyrin and its Ag, Zn, Co derivatives (H₂TOEPyP4, AgTOEPyP4, ZnTOEPyP4, CoTOEPyP4) were studied as potential anticancer drugs. A method that allows revealing the conformational state of DNA via the study of the effects of transition metal ions, such as Mn²⁺, on the intramolecular melting was used in the work. DNA melting was carried out in the presence of different stoichiometric concentrations of Mn²⁺ ions. The main melting parameters, the melting temperature and the melting range (T _m and ΔT), were calculated. The melting parameters of DNA isolated from tumor tissue differed from those of normal DNA. The melting parameters of DNA isolated from rats treated with porphyrins were close to the normal ones. The results showed that AgTOEPyP4 and ZnTOEPyP4 have a better antitumor effect than H₂TOEPyP4 and CoTOEPyP4.     

Differences between some properties of acetylated and nonacetylated forms of HMG1 protein

There are data suggesting that HMG1 protein may be involved in DNA replication. Recently we have found that only the acetylated form of the protein generates tetramers, stimulates the activity of DNA polymerase alpha (EC 2.7.7.7) (with activated DNA as a template) and forms a specific complex with it. This paper compares some properties of the acetylated and nonacetylated forms of HMG1 protein and shows that it is only the acetylated form which serves as a histone assembly factor, increases the melting temperature of poly d[(A-T)] and stimulates the activity of DNA polymerase alpha when histone H1-depleted chromatin is used as a template.     

Thermal denaturation of DNA in situ as studied by acridine orange staining and automated cytofluorometry

Thermal denaturation of nuclear DNA is studied in situ in individual cells or isolated cell nuclei by employing the property of the fluorochrome acridine orange (AO) to differentially stain native and denatured DNA and by using an automated flow-through cytofluorimeter for measurement of cell fluorescence. RNAse-treated cells, or cell nuclei, are heated, stained and measured while in suspension and AO-DNA interaction is studied under equilibrium conditions. Measurements are made rapidly (200 cells/sec); subpopulations of cells from a measured sample can be chosen on the basis of differences in their staining or light-scattering properties and analysed separately. DNA denaturation in situ is rapid; it approaches maximum during the first 5 min of cell heating. Divalent cations stabilize DNA against denaturation. At low pH the transition occurs at lower temperature and the width of the transition curves (‘melting profiles’) is increased. Decrease in ionic strength lowers the DNA melting temperature. This effect is much more pronounced in cells pretreated with acids under conditions known to remove histones. Histones thus appear to stabilize DNA in situ by providing counterions. At least four separate phases can be distinguished in melting profiles of DNA in situ; they are believed to indicate different melting points of DNA in complexes with particular histones. A decrease in cell (nuclear) ability to scatter light coincides with DNA melting in situ, possibly representing altered refractive and/or reflective properties of cell nuclei. Formaldehyde, commonly used to prevent DNA renaturation, is not used in the present method. The heat-induced alterations in nuclear chromatin are adequately stabilized after cell cooling in the absence of this agent. Cells heated at 60–85 °C exhibit increased total fluorescence after AO-staining, which is believed to be due to unmasking of new sites on DNA. This increase is neither correlated with DNA melting, nor with the presence of histones. Possibly, it reflects destruction of DNA superstructure maintained at lower temperatures by DNA associations with other than histone macromolecules (nuclear membrane).     

The HMG1 ta(i)le

We have studied structural changes in DNA/protein complexes using the CD spectroscopy, upon the interaction of HMG1-domains with calf thymus DNA at different ionic strengths. HMG1 protein isolated from calf thymus and recombinant HMG1-(A+B) protein were used. Recombinant protein HMG1-(A+B) represents a rat HMG1 lacking C-terminal acidic tail. At low ionic strength (15 mM NaCl) we observed similar behavior of both proteins upon interaction with DNA. Despite this, at higher ionic strength (150 mM NaCl) their interaction with DNA leads to a completely different structure of the complexes. In the case of HMG1-(A+B)/DNA complexes we observed the appearance of DNA fractions possessing very high optical activity. This could be a result of formation of the highly-ordered DNA structures modulated by the interaction with HMG1-domains. Thus the comparison studies of HMG1 and HMG1-(A+B) interaction with DNA show that negatively charged C-terminal tail of HMG1 modulates interaction of the protein with DNA. The striking difference of the behaviour of these two systems allows us to explain the functional role of multiple HMG1 domains in some regulatory and architectural proteins.     

The energetics of HMG box interactions with DNA: thermodynamics of the DNA binding of the HMG box from mouse sox-5

The energetics of the Sox-5 HMG box interaction with DNA duplexes, containing the recognition sequence AACAAT, were studied by fluorescence spectroscopy, isothermal titration calorimetry (ITC) and differential scanning calorimetry (DSC). Fluorescence titration showed that the association constant of this HMG box with the duplexes is of the order 4x10(7) M(-1), increasing somewhat with temperature rise, i.e. the Gibbs energy is -40 kJ mol(-1) at 5 degrees C, decreasing to -48 kJ mol(-1) at 32 degrees C. ITC measurements of the enthalpy of association over this temperature range showed an endothermic effect below 17 degrees C and an exothermic effect above, suggesting a heat capacity change on binding of about -4 kJ K(-1) mol(-1), a value twice larger than expected from structural considerations. A straightforward interpretation of ITC data in heat capacity terms assumes, however, that the heat capacities of all participants in the association reaction do not change over the considered temperature range. Our previous studies showed that over the temperature range of the ITC experiments the HMG box of Sox-5 starts to unfold, absorbing heat and the heat capacities of the DNA duplexes also increase significantly. These heat capacity effects differ from that of the DNA/Sox-5 complex. Correcting the ITC measured binding enthalpies for the heat capacity changes of the components and complex yielded the net enthalpies which exhibit a temperature dependence of about -2 kJ K(-1) mol(-1), in good agreement with that predicted on the basis of dehydration of the protein-DNA interface. Using the derived heat capacity change and the enthalpy and Gibbs energy of association measured at 5 degrees C, the net enthalpy and entropy of association of the fully folded HMG box with the target DNA duplexes was determined over a broad temperature range. These functions were compared with those for other known cases of sequence specific DNA/protein association. It appears that the enthalpy and entropy of association of minor groove binding proteins are more positive than for proteins binding in the major groove. The observed thermodynamic characteristics of protein binding to the A+T-rich minor groove of DNA might result from dehydration of both polar and non-polar groups at the interface and release of counterions. The expected entropy of dehydration was calculated and found to be too large to be compensated by the negative entropy of reduction of translational/rotational freedom. This implies that DNA/HMG box association proceeds with significant decrease of conformational entropy, i.e. reduction in conformational mobility.     

Structure-specific binding of the two tandem HMG boxes of HMG1 to four-way junction DNA is mediated by the A domain

We have investigated the nature of the "structure-specific" binding of the tandem A and B HMG boxes of high mobility group protein 1 (HMG1) to four-way junction DNA. AB didomain binding favours the open, planar form of the junction, as shown by reaction with potassium permanganate. Site-directed cleavage of the DNA by a 1, 10-phenanthroline-copper moiety attached to unique natural or engineered cysteine residues in the A or B domain shows that the two linked HMG boxes are not functionally equivalent in four-way junction binding. The A domain of the didomain binds to the centre of the junction, mediating structure-specific binding; the concave surface of the domain interacts with the widened minor groove at the centre, contacting one of the four strands of the junction, and the short arm comprising helices I and II and the connecting loop protrudes into the central hole. The B domain makes contacts along one of the arms, presumably stabilising the binding of the didomain through additional non-sequence-specific interactions. The isolated B domain can, however, bind to the centre of the junction. The preferential binding of the A domain of the AB didomain to the centre correlates with our previous finding of a higher preference of the isolated A domain than of the B domain for this structurally distinct DNA ligand. It is probably at least partly due to the higher positive surface potential in the DNA-binding region of the A domain (in particular to an array of positively charged side-chains suitably positioned to interact with the negatively charged phosphates surrounding the central hole of the junction) and partly to differences in residues corresponding to those that intercalate between bases in other HMG box/DNA complexes.     

Interaction of high mobility group proteins HMG 1 and HMG 2 with nucleosomes studied by gel electrophoresis

The binding of isolated high mobility group proteins HMG (1+2) with nucleosomes was studied using gel electrophoresis. The interaction of HMG (1+2) with mononucleosomes could be detected as a new discrete electrophoretic band with a decreased mobility only after cross-linking of HMG (1+2)-nucleosome complex by formaldehyde. Approximately two molecules of the large HMG proteins were bound per nucleosomal particle of a DNA length of 185 base pairs, lacking histones H1 and H5. Using the same techniques, no binding was observed with core particles of a DNA length of 145 base pairs.     

Model studies of DNA-protein interaction. I. Effect of aminocarboxylic and amide groups of amino acids on DNA thermostability and conformation

To elucidate interactions of amino-carboxylate dipole and amide group of amino acids with DNA, glycine and glutamine, concentration dependences of the melting curves and CD spectra of calf thymus DNA at low ionic strength (10(-4) M) Na-citrate have been studied. A considerable increase of the melting temperature delta t1/2 and a decrease of the temperature interval of melting delta t with the rise of glycine concentration were observed without changes in the CD spectrum. A comparison was made between the influence of dipolar glycine ion and isolated amino and carboxylate ions of ammonium acetate. The data obtained indicated the predominance of electrostatic interaction of glycine with DNA phosphates until the ligand concentration was approximately 0.6 M and, apparently, specific interactions of carboxylate ion with guanine at higher glycine concentrations. Destabilizing effect of glutamine on DNA at a concentration of 5.10(-3) M was observed, whereas at higher concentrations two-phase increase of delta t1/2 was shown. Small changes in DNA CD spectrum under the action of glutamine were registered. The comparison data for glutamine and acrylamide showed that DNA destabilizing effect was due to the amide group. The destabilizing effect of amide group can be explained by its interaction with the bases in single-stranded regions of DNA with the formation of two H-bonds. It is possible that the increase of DNA delta t1/2 is the result of the interaction of phosphates both with aminocarboxylate and amide groups of glutamine.     

The interaction of DNA with sperm-specific histones of the H1 family

We have studied the interactions of DNA with sperm-specific histones of the H1 family of sea urchin Strongylocentrotus intermedius, sea starfish Aphelasterias japonica and bivalve mollusk Chlamis islandicus using circular dichroism and DNA melting analysis. It was shown that echinoderm's sperm H1 protein has additional alpha-helical domains in its C-terminus and it demonstrates stronger DNA compaction. The differential melting curves of DNA-protein complexes have two peaks. The low temperature peak characterized the melting temperature of free DNA within the complex. The higher temperature peak characterizes the melting temperature of DNA bond to protein. DNA is found to be in the most stable state in the complexes with mollusk sperm H1 protein.     

DNA denaturation in situ. Effect of divalent cations and alcohols

Heat denaturation profiles of rat thymus DNA, in intact cells, reveal the presence of two main DNA fractions differing in sensitivities to heat. The thermosensitive DNA fraction shows certain properties similar to those of free DNA: its stability to heat is decreased by alcohols and is increased in the presence of the divalent cations Ca2+, Mn2+, or Mg2+ at concentrations of 0.1-1.0 mM. Unlike free DNA, however, this fraction denatures over a wide range of temperature, and is heterogeneous, consisting of at least two subfractions with different melting points. The thermoresistant DNA fraction shows lowered stability to heat in the presence of Ca2+, Mn2+, or Mg2+ and increased stability in the presence of alcohols. It denatures within a relatively narrow range of temperature, consists of at least three subfractions, and, most likely, represents DNA masked by histones. The effect of Ca2+, Mn2+, or Mg2+ in lowering the melting point of the thermoresistant DNA fraction is seen at cation concentrations comparable to those required to maintain gross chromatin structure in cell nuclei or to support superhelical DNA conformation in isolated chromatin (0.5-1.0 mM). It is probable that factors involved in the maintenance of gross chromatin organization in situ and/or related to DNA superhelicity also have a role in modulating DNA-histone interactions, and that DNA-protein interactions as revealed by conventional methods using isolated chromatin may be different from those revealed when gross chromatin morphology remains intact.     

Purification and characterization of DNA-dependent ATPase II from Escherichia coli.

A new DNA-dependent ATPase was isolated and purified from soluble extracts of Escherichia coli. This enzyme, called ATPase II, has a molecular weight of 86,000 and exists in a monomeric state. It degrades ATP (or dATP) to ADP (or dADP) and Pi in the presence of magnesium and requires a double-stranded polynucleotide as cofactor. A correlation between the efficiency as cofactor and the melting point of the polynucleotide has been found; the lower the melting temperature, the higher the stimulation of ATPase II. The enzyme binds to single-stranded DNA and poly[d(A-T)] copolymer, but not to the double-stranded circular DNA (Form I) of simian virus 40.     

Isolation of high-mobility-group proteins HMG1 and HMG2 in non denaturing conditions and comparison of their properties with those of acid-extracted proteins

We describe a method for isolation and purification of the chromosomal proteins HMG1 and HMG2 in non-denaturing conditions which overcomes the difficulties of the published methods concerning yield and purity. The method is based on salt extraction, selective precipitation with ammonium sulfate and DEAE-cellulose chromatography. All studied properties of these proteins (formation of protein tetramers, enhancement of micrococcal nuclease digestion of DNA and chromatin, and protection of 165-basepair DNA in chromatosome) differ significantly from the properties of HMG1 and 2 isolated under denaturing conditions.     

Spectral and physical characterization of the inverted terminal repeat DNA structure from adenoassociated virus 2

An oligodeoxynucleotide (ODN) that includes elements found in secondary structures at the 5'- and 3'- ends of adenoassociated virus 2 virion DNA was synthesized by ligation of three overlapping ODNs. The most stable secondary structure was calculated to be branched, with a 61 bp duplex stem, terminating in a three-way junction with 9 bp arms. The electrophoretic mobility of the ODN is slower than expected for normal duplex DNA of the same size, suggesting a bent or branched conformation. CD spectra indicate that the ITR structure is largely B form DNA, although there is a slight blue shift compared to the spectra of the isolated stem and loop elements. Thermal melting experiments indicate that the hairpin is significantly more stable than the isolated stem and loop elements. Singular value decomposition of UV spectra obtained as a function of temperature indicates that four species contribute to changes in the spectra upon denaturation, indicating that the melting is not a simple two-state process. Characterization of the branched ODN by differential scanning calorimetry permits estimation of the enthalpy of melting by a model-free analysis, yielding DeltaHcal= 614 kcal mol-1. This value agrees with the enthalpy computed for the most stable secondary structure.