Interaction of chromosomal proteins with BrdU substituted DNA as determined by chromatin-DNA competition.

Chromatin-DNA competition has been utilized to examine the general nature of chromosomal proteins interacting more strongly with BrdU substituted DNA. When chromatin is incubated with an excess of purified DNA, a portion of the chromosomal proteins will exchange to the purified DNA. These two complexes can then be separated on Metrizamide gradients due to their differing protein/DNA ratios. Using this technique we observe that most nonhistone chromosomal proteins will exchange to a competitor DNA, the extent of exchange being directly dependent upon the competitor DNA being present in excess. While essentially the same proteins will migrate to either unsubstituted or BrdU substituted DNA, the substituted DNA is found to be a quantitatively better competitor and its effectiveness as a competitor is directly related to the level of BrdU substitution.     

Optimal conditions and specificity of interaction of a distinct class of nonhistone chromosomal proteins with DNA.

A subclass of nonhistone chromatin proteins with high DNA affinity has been isolated from rat liver. The interaction of the isolated proteins with DNA in vitro was characterized utilizing a nitrocellulose filter binding technique. The temperature, time, concentration, ionic strength, and pH dependence were characterized. Optimal interaction was observed at 0.19 M naCl, pH 7.5 with a protein to DNA ratio of 13 (w/w). Equilibrium and kinetic competition experiments indicated that these proteins interact optimally with A-T rich and single-stranded DNA. The data also suggest that these proteins might affect the helixcoil transiton of DNA.     

Selective interaction of cytoskeletal proteins with liposomes

A protein kinase activity analogous to that found in interferon-treated HeLa cells is detectable in human plasma rich in platelets. This kinase activity is manifested by the phosphorylation of an endogenous Mr 72000 protein which could be conveniently assayed after partial purification on poly(G)-Sepharose. Here, we show that the protein kinase system in the plasma consists of at least 2 components. The protein kinase is found to be localised in the platelets whereas most of the substrate (the Mr 72000 protein) is found free in the plasma and a fraction of it associated with the surface of platelets.     

Selective interaction of tirapazamine with DNA bases and DNA

An electrochemical model has been used to study the reductive activation of the hypoxic cell cytotoxin tirapazamine (TPZ, 3-amino-1,2,4-benzotriazine-1,4-dioxide). Cyclic voltammetry and controlled potential electrolysis have been used to generate and study the 1-electron reduction product, the assumed biologically active species. Cyclic voltammetry of tirapazamine in dimethylformamide shows a quasi-reversible 1-electron reduction with the product showing a tendency to participate in a following chemical reaction. Controlled potential electrolysis to generate the 1-electron reduction product was unsuccessful due to the formation of a new redox-active species at less negative reduction potentials. However, the cyclic voltammetry of tirapazamine in the presence of E. coli DNA shows a decrease in the lifetime of the radical anion, signifying direct interaction with the DNA. The radical lifetime also decreased in the presence of adenine, thymine and guanine, but increased upon addition of cytosine and ribose. The study shows that cyclic voltammetry is an extremely useful tool for investigating the interaction between bio-reductive drugs and biological target molecules.     

Selective interaction of tirapazamine with DNA bases and DNA

An electrochemical model has been used to study the reductive activation of the hypoxic cell cytotoxin tirapazamine (TPZ, 3-amino-1,2,4-benzotriazine-1,4-dioxide). Cyclic voltammetry and controlled potential electrolysis have been used to generate and study the 1-electron reduction product, the assumed biologically active species. Cyclic voltammetry of tirapazamine in dimethylformamide shows a quasi-reversible 1-electron reduction with the product showing a tendency to participate in a following chemical reaction. Controlled potential electrolysis to generate the 1-electron reduction product was unsuccessful due to the formation of a new redox-active species at less negative reduction potentials. However, the cyclic voltammetry of tirapazamine in the presence of E. coli DNA shows a decrease in the lifetime of the radical anion, signifying direct interaction with the DNA. The radical lifetime also decreased in the presence of adenine, thymine and guanine, but increased upon addition of cytosine and ribose. The study shows that cyclic voltammetry is an extremely useful tool for investigating the interaction between bio-reductive drugs and biological target molecules.     

Association of eukaryotic DNA topoisomerase I with nucleosomes and chromosomal proteins.

A DNA topoisomerase activity is found to be associated with the nucleosomes released by the Staphylococcal nuclease digestion of HeLa nuclei. Such an association is found to be salt dependent. A number of criteria have established that this DNA topoisomerase activity is due to HeLa topo I (Liu, L. F. and Miller, K. G. (1980) Proc. Natl. Acad. Sci. USA 78, 3489-3491). A similar association has been demonstrated from the in vitro studies using purified mononucleosomes and eukaryotic DNA topoisomerase I. Nonhistone HMG proteins and histone H1 are found to stimulate topoisomerase activity in vitro and form tight complexes with eukaryotic DNA topoisomerase I. The intimate interactions of topoisomerase I with chromosomal proteins and nucleosomes may be an essential feature of the topoisomerase function in vivo.     

DNA sequence selection by tightly-bound nonhistone chromosomal proteins.

Extraction of chicken reticulocyte chromatin with 2.0 M NaCl removed 96% of chromosomal protein and yields two DNA components after dialysis and high-speed centrifugation. The bulk of chromosomal DNA (ca. 99%) is rendered free of protein, and is thus soluble in 10 mM Tris-HCl, pH 8.0. The other component (ca. 1%) displays a high protein/DNA ratio, and is insoluble in 10mM Tris-HCl, pH 8.0. These DNAs can be separated on the basis of their solubilities. Analysis of the reassociation kinetics with total chicken DNA of these DNAs reveals marked differences. Whereas total DNA and the soluble component (DNA-S) have rapidly reassociating components, the insoluble component (DNA-P) is devoid of these components, and is therefore composed completely of unique sequence DNA. Cot 1/2 values indicate that DNA-S is substantially depleted of some DNA-P sequences. We conclude that this segregation, as determined by tightly-bound nonhistone chromosomal proteins, selects a subset of total genomic DNA sequences, and suggests sequence-specific interaction between the tightly-bound nonhistones and DNA.     

5-Methylcytosine content of rat hepatoma DNA substituted with bromodeoxyuridine.

The aim of these experiments was to test whether incorporation of bromodeoxyuridine into DNA affects DNA methylation. Rat hepatoma (HTC) cells in culture were labeled for two generations with [14C]bromodeoxyuridine and [3H]thymidine to yield DNA which was 2.1, 20.6, 52.6, and 95.0% bromodeoxyuridine-substituted in the newly made strands. The DNA then was fractionated into highly repetitive, moderately repetitive, and single copy sequences. As determined by a comparison of 14C and 3H counts per min, the percentage of substitution with bromodeoxyuridine was found to be the same in each repetition class. The 5-methylcytosine content of each fraction was determined using high pressure liquid chromatography. It was found that bromodeoxyuridine, even at a level of substitution into newly mad DNA of 95%, has no effect on the 5-methylcytosine content of DNA. At all levels of bromodeoxyuridine substitution, highly repetitive DNA has slightly more 5-methylcytosine (3.0% of total cytosine) than does single copy DNA or moderately repetitive DNA (2.3%). The 5-methylcytosine content of whole HTC DNA is the same as that of rat liver DNA (2.4%).     

Conformational studies of two non-histone chromosomal proteins and their interactions with DNA.

The conformational properties of two non-histone chromosomal proteins (high-mobility-group proteins 1 and 2) have been studied by spectroscopic methods. The interaction of high-mobility-group protein 1 with DNA has also been studied. 1. Circular dichroism results indicate that in the presence of salt both proteins are 40-50% helical between pH 1 and 9. Above pH 9 denaturation takes place. In the absence of salt the proteins denature below pH 4. 2. Nuclear magnetic resonance spectra show the presence of ring-current shifted peaks and perturbed aromatic resonances, demonstrating that the helix formation is accompanied by specific tertiary folding. 3. Nuclear magnetic resonance spectra of compelxes between high mobility group protein 1 and DNA demonstrate that a low ionic strength a portion of the molecule rich in lysine and containing all the aromatic residues is bound to DNA, whilst a more acidic region of the chain remains free from the DNA.     

The interaction of substituted and rigidly linked diquinolines with DNA

Viscometric measurements with circular and sonicated rodlike DNA fragments were used to explore whether ring substituents or conformationally restricted linkers promote bifunctional intercalation amongst a series of binuclear 4-aminoquinolines bridged via their 4-amino group. We find that ligands comprising unsubstituted quinolines and piperazine or pyrazole linkages bisintercalate. Quinoline-substituted alkyl-linked dimers intercalate in either a mixed monofunctional-bifunctional mode or bind with only one of their chromophores intercalated depending on the nature of the substituents. Equilibrium dialysis measurements show that the binding affinity for calf thymus DNA of the compounds studied ranges from (1.2-12) . 10(4) M-1 in buffer of ionic strength 0.1. Both co-operative and antico-operative binding isotherms were obtained and there is evidence for a second binding mode for the piperazine-linked diquinoline at saturating binding levels. For this compound the high-affinity association constant decreases with increasing ionic strength, 3.4 cations being released per bound ligand molecule. Partition dialysis measurements with DNAs of differing base composition indicate that the compounds studied are either AT selective or sequence neutral depending on ligand structure. For example, the pyrazole linker imparts a marked specificity for binding to AT-rich DNA, whereas the piperazine linker does not. Kinetic measurements using the surfactant-sequestration method reveal that DNA-diquinoline complexes dissociate very rapidly by complex mechanisms with rate constants greater than 100 s-1 in buffer of ionic strength 0.1.     

The interaction of DNA mismatch repair proteins with human exonuclease I.

Exonucleolytic degradation of DNA is an essential part of many DNA metabolic processes including DNA mismatch repair (MMR) and recombination. Human exonuclease I (hExoI) is a member of a family of conserved 5' --> 3' exonucleases, which are implicated in these processes by genetic studies. Here, we demonstrate that hExoI binds strongly to hMLH1, and we describe interaction regions between hExoI and the MMR proteins hMSH2, hMSH3, and hMLH1. In addition, hExoI forms an immunoprecipitable complex with hMLH1/hPMS2 in vivo. The study of interaction regions suggests a biochemical mechanism of the involvement of hExoI as a downstream effector in MMR and/or DNA recombination.     

Specific interaction between mouse liver non-histone chromosomal proteins and mouse DNA demonstrated by a sequential DNA-protein binding procedure.

The binding of mouse liver chromosomal proteins to DNA has been investigated using the nitrocellulose filter binding technique. Careful purification of the DNA involving nuclease S1 digestion and prefiltration through a nitrocellulose filter is used to reduce background binding in the absence of protein to less than 1%. Procedures involving direct binding of protein to labeled DNA, competition of binding of labeled DNA by unlabeled DNA, and dissociation of DNA . protein complexes with time do not indicate significant preference for binding to mouse DNA relative to Escherichia coli DNA. This specificity is demonstrated much more clearly by a novel type of procedure, which we call a sequential binding procedure. In this procedure non-specific binding proteins are sequestered by incubation with an excess of unlabeled E. coli DNA prior to addition of labeled DNA. Under these conditions, labeled mouse DNA is bound to filters to a 3- to 4-fold greater extent than labeled E. coli DNA.     

Selective DNA bending by a variety of bZIP proteins.

We have investigated DNA bending by bZIP family proteins that can bind to the AP-1 site. DNA bending is widespread, although not universal, among members of this family. Different bZIP protein dimers induced distinct DNA bends. The DNA bend angles ranged from virtually 0 to greater than 40 degrees as measured by phasing analysis and were oriented toward both the major and the minor grooves at the center of the AP-1 site. The DNA bends induced by the various heterodimeric complexes suggested that each component of the complex induced an independent DNA bend as previously shown for Fos and Jun. The Fos-related proteins Fra1 and Fra2 bent DNA in the same orientation as Fos but induced smaller DNA bend angles. ATF2 also bent DNA toward the minor groove in heterodimers formed with Fos, Fra2, and Jun. CREB and ATF1, which favor binding to the CRE site, did not induce significant DNA bending. Zta, which is a divergent member of the bZIP family, bent DNA toward the major groove. A variety of DNA structures can therefore be induced at the AP-1 site through combinatorial interactions between different bZIP family proteins. This diversity of DNA structures may contribute to regulatory specificity among the plethora of proteins that can bind to the AP-1 site.     

DNA interaction with low molecular weight ligands of different structure. III. DNA complexes with distactins

The DNA complexes with distactins have been investigated by means of spectrophotometry, viscosimetry and flow birefringence methods. The distactins are actinocin's derivatives containing in the 1,9 positions of the phenoxazone moiety oligopyrrolcarboxamide groups (like those of distamycin A), which have from one to three fragments of 1-methyl-4-amino-2-pyrrolic acid. The mode of DNA-distactins binding in water solution depends on the quantity of the methylpyrrole rings in the oligopeptide groups. The ligand with oligopeptide groups containing three methylpyrrole rings joins the DNA double helix only from outside by means of oligopeptide groups. The compounds with one and two methylpyrrole rings form two kinds of complexes with DNA: external binding and intercalation. In the latter case both chromophore and methylpyrrole fragments, interact with DNA.     

Organization of 5-methylcytosine in chromosomal DNA

The 5-methylcytosine residues of L-cells have been labeled with [methyl-3H]-L-methionine and their chromatin localization studied using deoxyribonucleases. The kinetics of micrococcal nuclease digestion showed that the methylated cytosine residues are concentrated within regions resistant to nuclease digestion and preferentially missing from those regions between nucleosomes which are nuclease sensitive. Using DNA hybridization kinetic analysis, it is shown that 5-methylcytosine is abundant in highly repeated sequences but is also present in middle repetitive and unique sequence DNA.     

Effects on chromosomal proteins in sister chromatid differentiation by incorporation of 5-bromodeoxyuridine into DNA

When fixed metaphase chromosomes of human lymphocytes grown in the presence of BrdUr for two cell cycles were stained with amino group-specific 2-methoxy-2,4-diphenyl-3(2H)furanone (MDPF) after a previous extraction of DNA, sister chromatids showed a light-independent differential staining. Although more faintly differential, a similar staining pattern being just the reverse of the DNA-specific DAPI pattern was obtained without prior removal of DNA. We conclude that the chromatid containing bifilarly BrdUr-substituted DNA has a higher protein content, at least after fixation, than the chromatid containing unifilarly BrdUr-substituted DNA. Possibly, a higher degree of BrdUr substitution leads to a tighter binding of chromosomal proteins. In line with this suggestion we found a markable difference in DNA extractability of BrdUr-containing and normal cytological preparations.