Cytofluorometric studies on conformation of nucleic acids in situ. I. Restriction of acridine orange binding by chromatin proteins.

Binding of the fluorochrome acridine orange (AO) to nucleic acids in situ is studied by automated cytofluorometry in two differentiating cell systems: Friend virus-transformed murine erythroleukemia induced to differentiate by dimethyl sulfoxide, and phytohemagglutinin-stimulated human lymphocytes. The specificity of the stain for deoxyribonucleic acid is discussed on the basis of data obtained by cell treatment with nucleases. Evidence is presented that in the case of Friend leukemia cells, but not phytohemagglutinin-stimulated lymphocytes, a significant change in the number of AO-intercalating sites in DNA occurrs during differentiation. These results suggest that changes in nuclear chromatin occurring during cell differentiation may be correlated, in some but not all systems, with changes in accessibility of DNA in situ to intercalating dyes. The role of divalent cations, especially Mg2+, in the conformation of nuclear chromatin and in modulation of the accessibility of nucleic acids to AO is discussed. The method provides a tool for the study of nucleic acid-protein interaction in situ, and in some cell systems it may be applicable as a marker for recognition of cell transformation, differentiation or neoplasia.     

Microsomal activation of estrogens and binding to nucleic acids and proteins.

Natural and synthetic estrogens can be activated by rat liver microsomes to bind covalently to polyguanylic acid, single-stranded DNA, nucleotides and proteins. Incubation of polyG, estrone and liver microsomes (0.5 nmole cytochrome P-448 or P-450) from 3-methylcholanthrene-induced, phenobarbital-induced or control rats showed that the former microsomes gave better $\text{net}$ binding of estrogens to polyG than the other two. Estradiol incubated with 3MC-induced microsomes did bind to DNA but marginally to polyG. Mestranol and estrone sulfate, both constituents of oral contraceptive formulations, bound to polyG whereas progesterone and cholesterol did not bind. We present also preliminary data on the characterization of estrogen-nucleic acid interactions using nucleases, proteinase K and high-pressure liquid chromatography.     

Physical studies of the interaction between the Escherichia coli DNA binding protein and nucleic acids.

The interaction of nucleic acid with the Escherichia coli DNA-binding protein has been studied by fluorescence emission spectroscopy and sedimentation velocity analysis. The protein binds to single-strand DNA with an apparent equilibrium dissociation constant of 2 X 10(-9). It binds to the homopolymers poly (dA) and poly (dT) slightly more tightly, but has a larger apparent equilibrium dissociation constant to poly (dC). The protein also binds tightly to ribohomopolymers and to tRNA, but not to duplex DNA. By the use of defined-length oligonucleotides, it has been shown that the protein binds to DNA in a highly cooperative manner. The extent of cooperativity is seen as the difference in binding between an isolated monomeric protein molecule bound to DNA and two or more molecules binding to contiguous sites.     

Interactions of molecules with nucleic acids. I. An algorithm to generate nucleic acid structures with an application to the B-DNA structure and a counterclockwise helix.

An algorithm is developed that enables the routine determination of backbone conformations of nucleic acids. All atomic positions including hydrogen are specified in accord with experimental bond lengths and angles but with theoretically determined conformational angles. For two Watson-Crick base pairs at a separation of 3.38 Å, and perpendicular to a common helical axis, minimum energy configurations are found for all 10 combinations at helical angles of α ～ 36°–38°, corresponding to the B-DNA structure with C(2′)-endo sugar puckers. Backbone configurations exist only within the range 35.5° ? α ? 42°, which suggests the origin of the 10-fold helix. Calculated stacking energies for the B-DNA structure increases for each of the clustered groups of base pairs: G·C with G·C, G·C with A·T, and A·T with A·T, and they are in approximate agreement with experimental observations. The counter-clockwise helix is examined, and physically meaningful structures are found only when the helical axes of successive base pairs are disjointed.     

Theoretical studies on protein-nucleic acid interactions. I. Interaction of positively charged amino acids with nucleic acid fragments

Coulombic interactions between the side chains of charged amino acids (Arg⁺, Lys⁺, and His⁺) and negatively charged phosphate groups of nucleic acid fragments have been studied theoretically. Diribose monophosphate and dideoxyribose monophosphate are chosen as model systems for single-stranded RNA and DNA, respectively. The interaction energies have been calculated by second-order perturbation theory using simplified formulas for individual terms. The interaction energy in this formalism is a sum of electrostatic, polarization, dispersion, and repulsive energies. Our results show that about 90% of the total interaction energy is contributed by the electrostatic term alone. Contribution from the repulsive term exceeds that from the dispersion term. Calculated interaction energies suggest that Lys⁺ and His⁺ form more stable complexes with RNA than with single-stranded DNA. On the other hand, Arg⁺ has a higher affinity for DNA than for RNA. The affinity of nucleic acids for the three amino acids is in the order Lys⁺ > His⁺ > Arg⁺. Further, the basic amino acid residues form more stable complexes with A-DNA than with B-DNA. The role of the Coulombic interactions in the specific recognition of nucleic acids by proteins is discussed.     

Cooperative binding of m-AMSA to nucleic acids.

The equilibrium binding of the antitumor agent m-AMSA (4'-(9-acridinylamino) methane-sulfon-m-ansidide) has been examined by optical methods. These studies which have focused on the low bound drug concentrations (r values less than 0.02, base pairs) reveal m-AMSA to bind calf thymus DNA in a highly cooperative manner as indicated by the initial positive slope of the Scatchard plot. In contrast, the studies on the parent 9-aminoacridine under identical conditions demonstrate that this compound binds DNA in a noncooperative (neighbor exclusion) manner. The positive cooperative binding phenomenon of m-AMSA is probed as a function of ionic concentration and shown to exist over the range of salt concentrations examined (0.01 to 0.1 M); however, the magnitude of the cooperative binding is altered. This observation of cooperativity is consistent with earlier studies on biologically active compounds and may be related to such binding parameters as binding sequence selectivity and/or structural perturbations to the DNA structure.     

Electrophoretic transfer of proteins and nucleic acids from slab gels to diazobenzylozymethyl cellulose or nitrocellulose sheets

A new electrophoretic technique for transferring proteins and nucleic acids from either polyacrylamide or agarose slab gels has been developed as an alternative to blotting. The method is fast, efficient and versatile. Efficient transfer of DNA to both nitrocellulose membrane and diazobenzyloxymethyl cellulose as well as efficient transfer of proteins to diazobenzyloxymethyl cellulose is demonstrated.     

Separation of nucleic acids and chromatin proteins by hydrophobic interaction chromatography

A procedure by which chromatin proteins (histones and non-histones) can be rapidly separated from nucleic acids by hydrophobic interaction chromatography is described. The procedure is carried out under non-rigorous conditions that must be assumed to induce little irreversible change in the biological properties of most proteins. More than 90% (w/w) of the chromatin proteins can be retained by hydrophobic interaction while nucleic acids pass quantitatively through the columns. By gradient elution of the columns the histones can be divided into fractions containing H1, H2A/H2B and H3/H4, and at the same time a subfractionation of the non-histone proteins is obtained. Protein recovery depends on the type of column used, but exceeds 80% (w/w) with even the most strongly binding hydrophobic matrix investigated.     

Interactions of aromatic residues of proteins with nucleic acids. Fluorescence studies of the binding of oligopeptides containing tryptophan and tyrosine residues to polynucleotides.

The binding of oligopeptides of general structure Lys-X-Lys (where X is an aromatic residue) to several polynucleotides has been studied by fluorescence spectroscopy. Two types of complexes are formed, both involving electrostatic interactions between lysyl residues and phosphate groups as shown by the ionic strength and pH dependence of binding. The fluorescence quantum yield of the first complex is identical with that of the free peptide. The other complex involves a stacking of the nucleic acid bases with the aromatic amino acid whose fluorescence is quenched. Fluorescence data have been quantitatively analyzed according to a model involving these two types of complexes. Association constants and the size of binding sites have been determined. Stacking interactions are favored in single-stranded polynucleotides as compared to double-stranded ones. A short oligopeptide such as Lys-X-Lys is thus able to distinguish between single-stranded and double-stranded nucleic acids. Fluorescence results are compared to those obtained by proton magnetic resonance and circular dichroism.     

Folding energetics of ligand binding proteins. I. Theoretical model

Heat capacity curves as obtained from differential scanning calorimetry are an outstanding source for molecular information on protein folding and ligand-binding energetics. However, deconvolution of C(p) data of proteins in the presence of ligands can be compromised by indeterminacies concerning the correct choice of the statistical thermodynamic ensemble. By convent, the assumption of constant free ligand concentration has been used to derive formulae for the enthalpy. Unless the ligand occurs at large excess, this assumption is incorrect. Still the relevant ensemble is the grand canonical ensemble. We derive formulae for both constraints, constancy of total or free ligand concentration and illustrate the equations by application to the typical equilibrium Nx <=> N + x <=> D + x. It is demonstrated that as long as the thermodynamic properties of the ligand can be completely corrected for by performing a reference measurement, the grand canonical approach provides the proper and mathematically significantly simpler choice. We demonstrate on the two cases of sequential or independent ligand-binding the fact, that similar binding mechanisms result in different and distinguishable heat capacity equations. Finally, we propose adequate strategies for DSC experiments as well as for obtaining first estimates of the characteristic thermodynamic parameters, which can be used as starting values in a global fit of DSC data.     

Models of interaction between nucleic acids and proteins. Hydrogen bonding of arginine with nucleic acid bases, phosphate groups and carboxylic acids.

Complex formation between the side chain of arginine and nucleic acid bases has been investigated by proton magnetic resonance in dimethylsulfoxide. Simultaneous formation of two hydrogen bonds leads to a selectivity of arginine interaction towards cytosine and guanine. A comparison is made of the interaction of arginine side chain with nucleic acid bases, phosphate and carboxylate anions. It is shown that interaction between carboxylate and arginine is stronger than between phosphate and arginine. These results are discussed with respect to the selective recognition of nucleic acid bases by arginine side chains and by the arginyl-glutamyl ion pair which could form in proteins interacting with nucleic acids.     

The interaction of chromium with nucleic acids

Native and denatured calf thymus DNA, and homopolyribonucleotides were compared with respect to chromium and protein binding after an in vitro incubation with rat liver microsomes, NADPH, and chromium(VI) or chromium(III). A significant amount of chromium bound to DNA when chromium(VI) was incubated with the native or the denatured form of DNA in the presence of microsomes and NADPH. For both native and denatured DNA the amount of protein bound to DNA increased with the amount of chromium bound to DNA. Denatured DNA had much higher amounts of chromium and protein bound than native DNA. There was no interaction between chromium(VI) and either form of DNA in the absence of the complete microsomal reducing system. The binding of chrornium(III) to native or denatured DNA was small and relatively unaffected by the presence of microsomes and NADPH. The binding of chromium and protein to polyriboadenylic acid (poly(A)), polyribocytidylic acid (poly(C), polyri-boguanylic acid (poly(G)) and polyribouridylic acid (poly(U)) was determined after incubation with chromium(VI) in the presence of microsomes and NADPH. The magnitude of chromium and protein binding to the ribo-polymers was found to be poly(G) ? poly(A) ? poly(C) ? poly(U). These results suggest that the metabolism of chromium(VI) is necessary in order for chromium to interact significantly with nucleic acids. The metabolically-produced chromium preferentially binds to the base guanine and results in DNA-protein cross-links. These findings are discussed with respect to the proposed scheme for the carcinogenicity of chromium(VI). Keywords: DNA-protein cross-links — Chromium-guanine interaction-Microsomal reduction of chromate     

The flexibility of the nucleic acids: (III). The interaction of an aliphatic diamine, putrescine, with flexible B-DNA

A theoretical modelling of the interaction of putrescine (H3+N-(CH2)4-(+NH3) with DNA is carried out, introducing two new features which make the simulation of this interaction considerably more realistic. Firstly, the DNA to which putrescine is bound is fully flexible and thus able to respond to the distorting influence of the ligand. Secondly, the effect of changing the ratio of DNA base pairs per bound ligand is explicitly modelled. In this way, we have been able to confirm the experimentally known preference of putrescine binding with AT base pairs in B-DNA, but we also show, through the new features introduced, that the nature of the binding site of the ligand and the resulting impact on DNA conformation is strongly modified by the ligand binding density.