Studies on DNA bound to histones in chromatin]

Regions of DNA protected by histones against the action of DNAse 1 in the chromatin were isolated. Such DNA fragments ("subhistones" DNA) have 80% double helix structure, their nucleotide composition is close to that of total DNA, and their sedimentation constant is within the range of 2-2.7S for completely denatured molecules. Kinetics of renaturation of "subhistone" DNA was studied: within a wide range of Cot values, renaturation curves of total and "subhistone" DNA are almost identical. According to the data on hybridization with nuclear d-RNA, "subhistone" DNA is transcribed in the cell. The data obtained witness for uniform character of distribution of histones along the DNA chain in the chromatin. DNA sites which are active in RNA synthesis seem to be bound to histones as well as the non-active ones. No significant difference was found in the hybridization of "subhistone" DNA from rat liver and thymus with ibver nuclear RNA.     

Determination of the rate of renaturation of modified DNA by fluorescence depolarization

Fluorescence depolarization was used to measure the rate of renaturation of T2 DNA, which had been modified by chloroacetaldehyde. Rates were measured on DNA samples with 5–15% of the base pairs modified and were found to agree with rates determined by DNA absorbance kinetics at 260 nm. The renaturation rate of a modified T2 DNA was unchanged in the presence of a ninefold abundance of unlabeled calf thymus DNA.     

The influence of glucosylation on the renaturation rate of T4 phage DNA

The kinetic complexity of T4 phage DNA with different degrees of glucosylation was determined by studies of DNA renaturation. It was found that this parameter decreased with decreasing degree of glucosylation, and that the kinetic complexity of non-glucosylated DNA was in agreement with the known genome size of T4 phage. This observation indicates that no significant correction for differences in GC content is necessary in the determination of genome sizes from renaturation data.     

p53 binds single-stranded DNA ends through the C-terminal domain and internal DNA segments via the middle domain.

We have previously reported that wild-type p53 can bind single-stranded (ss) DNA ends and catalyze renaturation of ss complementary DNA molecules. Here we demonstrate that p53 can also bind to internal segments of ss DNA molecules via a binding site (internal DNA site) distinct from the binding site for DNA ends (DNA end site). Using p53 deletion mutants, the internal DNA site was mapped to the central region (residues 99-307), while the DNA end site was mapped to the C-terminal domain (residues 320-393) of the p53 protein. The internal DNA site can be activated by the binding of ss DNA ends to the DNA end site. The C-terminal domain alone was sufficient to catalyze DNA renaturation, although the central domain was also involved in promotion of renaturation by the full-length protein. Our results suggest that the interaction of the C-terminal tail of p53 with ss DNA ends generated by DNA damage in vivo may lead to activation of non-specific ss DNA binding by the central domain of p53.     

Fractionation of calf thymus DNA based on its interaction with homologeous f1 histone. Melting curves of the obtained fractions

Calf thymus DNA fractions were obtained by precipitation with the homologeous f₁ histone and their melting curves were investigated. An increase of the melting temperatures of DNA remaining in the supernatants was observed. Within the range of 5–50 % of the DNA precipitated the melting temperatures and the melting intervals of DNA in the sediment remained constant. The obtained values /38 mole % GC, ΔT_m = 7.0°C/ suggest that DNA found in the precipitates corresponds to the main calf thymus DNA. Despite its heterogeneity this group of DNA molecules does not undergo fractionation using f₁ histone. We assume that the molecules of the main DNA all contain specific areas to which the f₁ histone attaches in our experimental conditions. The main DNA molecules, regardless of their base composition, seem to contain these specific areas in amounts causing equal precipitation probability. They seem to differ in this respect from some GC rich fractions, possibly those of satellite DNA.     

Quantitative Aspects of Deoxyribonucleic Acid Renaturation: Base Composition, State of Chromosome Replication, and Polynucleotide Homologies

The base composition of a deoxyribonucleic acid (DNA) sample affects its intrinsic rate of renaturation. In agreement with the information of Wetmur and Davidson, it was established that high guanosine plus cytosine (GC) DNA renatures faster than expected from analytical measurement of its molecular weight. A calculated correction factor of 1.8% of the observed C(0)t(.5) is required for every mole per cent GC difference from 51% GC. The correction factor is now established in the range of 32 to 65% GC. Renaturation of DNA mixtures prepared from pairs of organisms has been studied. When no similarity existed between the two organisms, the observed C(0)t(.5) of the mixture was the sum of the independently determined C(0)t(.5) values. Lack of additivity was correlated with similarities in polynucleotide sequence of the reassociating DNA molecules. A quantitative relationship was formulated to relate C(0)t(.5) values of renatured DNA mixtures to per cent binding ("homology"). Finally, it was demonstrated that DNA prepared from log-phase cells renatures faster than stationary-phase DNA and also departs from theoretical second-order kinetics.     

Numerical simulations of Raman spectra of guanine-cytosine Watson-Crick and protonated Hoogsteen base pairs

Large changes in the Raman spectra of calf thymus DNA are observed upon lowering the pH. In order to gain a better insight into these effects, several simulations of the Raman spectra of the guanine-cytosine (GC) Watson-Crick and Hoogsteen base pairs are performed. By comparing the Raman bands of GC base pairs in calf thymus DNA at high and low pH with the theoretical simulations of GC base pairs, it is found that the intensity changes in the theoretical bands located between 400 and 1000 cm(-1) are small compared to the experimental ones. The behavior of the cytosine band at 1257 cm(-1) upon lowering the pH is not reproduced in the GC theoretical spectra. The bands located above 1300 cm(-1) in the theoretical spectra display intensity changes that are similar to those found for GC base pairs in calf thymus DNA spectra.     

ATP-independent renaturation of complementary DNA strands by the mutant recA1 protein from Escherichia coli

In an effort to clarify the requirement for ATP in the recA protein-promoted renaturation of complementary DNA strands, we have analyzed the mutant recA1 protein which lacks single-stranded DNA-dependent ATPase activity at pH 7.5. Like the wild type, the recA1 protein binds to single-stranded DNA with a stoichiometry of one monomer per approximately four nucleotides. However, unlike the wild type, the mutant protein is dissociated from single-stranded DNA in the presence of ATP or ADP. The ATP analogue adenosine 5'-O-3' (thiotriphosphate) appears to stabilize the binding of recA1 protein to single-stranded DNA but does not elicit the stoichiometry of 1 monomer/8 nucleotides or the formation of highly condensed protein-DNA networks that are characteristic of the wild type recA protein in the presence of this analogue. The recA1 protein does not catalyze DNA renaturation in the presence of ATP, consistent with the dissociation of recA1 protein from single-stranded DNA under these conditions. However, it does promote a pattern of Mg2+-dependent renaturation identical to that found for wild type recA protein.     

Characterization of enantioselective binding of racemic natural tetrahydropalmatine to DNA by chromatographic methods

A racemate from natural product, tetrahydropalmatine (THP), was characterized on its enantioselective binding to DNA by the chromatographic methods including microdialysis/HPLC, centrifugal ultrifiltration/HPLC and immobilized DNA affinity chromatography. It was found that its (+)-enantiomer was preferential to binding on B-form duplex DNA including calf thymus DNA, AT and GC sequence oligo DNA, as well as triplex oligo DNA. The binding constants of the THP enantiomers to ct-DNA were determined with the methods of microdialysis/HPLC and frontal affinity chromatography. In addition, the DNA structural preference of either enantiomer was evaluated with the chromatographic methods.     

Influence of formamide on the thermal stability of DNA

The utility of formamide in the denaturation and renaturation of DNA has been examined. The melting temperature of duplex DNA is lowered by 0·6°C per per cent formamide. The depression of melting temperature is independent of the GC content. Formamide also increases the width of the thermal transition. Upto 30%, it does not affect the rate of DNA reassociation     

Dictamnine,an alkaloid which crosslinks DNA in the presence of ultraviolet light

In the presence of ultraviolet light, the furoquinoline alkaloid, dictamnine, caused calf thymus DNA to become easily renaturable. The effect was less pronounced than for the furocoumarin, 8-methoxypsoralen. Ease of renaturation is evidence of the formation of interstrand crosslinks in DNA. The mechanism of crosslink formation by this alkaloid may be like that of 8-methoxypsoralen.     

Molecular hybridization between rat liver deoxyribonucleic acid and complementary ribonucleic acid

RNA (cRNA) was synthesized in vitro on a template of rat liver DNA and its hybridization with rat liver DNA was studied by using the nitrocellulose-filter method. Sonication of the DNA diminished its apparent capacity to hybridize with RNA by about 50%. This is not due to cross-linkage of DNA molecules, because it could be shown that less than 2% of the sonicated DNA was cross-linked. The effect is due instead to the small size of the sonicated DNA molecules. Below a single-stranded molecular weight of 5×10⁵ the DNA showed a progressive loss of capacity to hybridize with decrease in molecular weight. Evidence is presented suggesting that the apparently diminished capacity of the DNA to hybridize is due to loss of hybridized DNA from the membrane filters. When cRNA at concentrations of up to 25μg/ml is annealed with sonicated total DNA, an apparent hybridization saturation value is found at which about 2.5% of the DNA is hybridized with RNA. Increasing the cRNA concentration tenfold brought about the hybridization of a second component of the DNA approximately equal in amount to the first. The renaturation of rat liver DNA was studied by measuring the fall in the extinction at 260nm and two different components of renaturation were observed within the reiterated fraction of DNA. By hybridizing cRNA with different fractions of rat DNA the two components of the hybridization curve are shown to correspond to the two components of the renaturation curve. The conclusion is drawn that at a cRNA concentration of 250μg/ml most of the reiterated fraction of rat liver DNA is hybridized after annealing for 16h under standard conditions (0.30m-sodium chloride–30mm-sodium citrate at 65°C). Even with such a high cRNA concentration little or no hybridization of the slowly renaturing DNA fraction occurs. It is suggested that the most highly reiterated DNA component is poorly transcribed in vitro.     

Specific thymic peptides-DNA interaction. Correlation with the possible stereochemical kinking scheme of DNA

Low molecular weight peptides from calf thymus cause a strong dose-dependent stabilization of the DNA. The strength of DNA-peptide interaction is pH-dependent and decreases rapidly above pH 6.5. Moreover the complete kinetics of DNA denaturation and renaturation demonstrates that the peptide fraction increases significantly the DNA renaturation mostly at low temperature, showing that the interaction DNA-thymic effector helps the recombination of complementary DNA segments. The DNA stabilization rate by the peptide fraction is comparable to that obtained by means of high concentration of histones or synthetic polycationic peptides. However, the lack of basic amino acids in the peptide structure is not in favor of strong electrostatic interactions and implies a specific binding of peptide to DNA. The possible correlation of the specific thymic peptides-DNA interaction with the stereochemical kinking scheme of DNA is discussed.     

Renaturation effects of cis and trans platinum II and IV compounds on calf thymus deoxyribonucleic acid.

The effects of cis dichlorodiammine platinum [cis Pt(II)], trans dichlorodiammine platinum (trans Pt(II)], cis tetrachlorodiammine platinum [cis Pt(IV)], trans tetrachlorodiammine platinum [trans Pt(IV)], and ethylenediaminedichloride platinum [Pt(II)en] on the absorption spectra, and thermal hyper- and hypochromicity of calf thymus DNA were investigated. Platinum-induced renaturation was studied as one parameter of interstrand cross-linking. Based on a DNA cross-linking hypothesis, the tumor-inhibitory platinum compounds cis Pt(II), cis Pt(IV) and Pt(II)en would be expected to induce renaturation following thermal denaturation, whereas the ineffective drugs, trans Pt(II) and trans Pt(IV) would not. All five bind to DNA in such a way as to induce renaturation. However, cis Pt(IV) requires at least a 3- to 4-fold longer incubation time than is required by the other compounds to form the coordination bonds necessary for renaturation. Maximum renaturation with all compounds was observed at a molar Pt/base ratio of 0.05 except cis Pt(IV), with which it was 0.25. The rate of the formation of the platinum-coordinated cross-links by fresh cis Pt(II) suggests two reactions or types of reactions occur. The first is rapid and destabilizes the DNA helix, whereas the second is slow and responsible for renaturation following thermal denaturation. These results suggest that cis Pt(IV) may be activated cellularly and that cross-linking is not the primary mechanism of action of the tumor-inhibitory platinum compounds.     

Deoxyribonucleic acid binding studies on several new anthracycline antitumor antibiotics. Sequence preference and structure--activity relationships of marcellomycin and its analogues as compared to adriamycin.

The deoxyribonucleic acid (DNA) binding characteristics of adriamycin and several new anthracycline glycosides, including marcellomycin, aclacinomycin, rudolfomycin, musettamycin, and pyrromycin, have been studied. The fluorescence spectra were determined for all six anthracyclines, and the fluorescence quenching effects caused by interactions with the natural DNAs poly(dAdT)--poly(dAdT) and poly(dGdC) were characterized. Binding parameters were determined by Scatchard analyses of results obtained by spectrofluorometric titrations of anthracyclines with DNA. Consistent with earlier structure--activity relationship studies of nucleic acid synthesis inhibitory effects, the results demonstrate a correlation between the length of the glycosidic side chain and DNA binding affinity. In addition, the sugar residue 2-deoxyfucose appears to confer greater DNA binding ability than do the sugars rednosamine and cinerulose when present in the terminal position of the glycosidic side chain, also in agreement with earlier studies. The sequence preference of anthracycline--DNA interaction has been examined by using DNAs of varying GC content, including the naturally occurring calf thymus DNA (43% GC), Clostridium perfringens DNA (28% GC), and Micrococcus luteus DNA (72% GC) and the synthetic double-stranded copolymers poly(dGdC)--poly(dGdC) and poly(dAdT)--POLY(DAdT). The results demonstrate that although adriamycin shows an absolute requirement for GC sequences for DNA binding, marcellomycin and its analogues showed no such sequence requirement. Furthermore, an AT preference for DNA binding was demonstrated with marcellomycin and its analogues.     

The DNA of CAENORHABDITIS ELEGANS

Chemical analysis and a study of renaturation kinetics show that the nematode, Caenorhabditis elegans, has a haploid DNA content of 8 x 10(7) base pairs (20 times the genome of E. coli). Eighty-three percent of the DNA sequences are unique. The mean base composition is 36% GC; a small component, containing the rRNA cistrons, has a base composition of 51% GC. The haploid genome contains about 300 genes for 4S RNA, 110 for 5S RNA, and 55 for (18 + 28)S RNA.     

Kinetics of renaturation of denatured DNA. II. Products of the reaction

The structure of renatured T4 DNA has been studied by CsCl density-gradient centrifugation. It has been found that the products of the reaction differ, depending on the method used for denaturation of the DNA. If denaturation is carried out without taking precautions to prevent chain degradation, for example, by heat, the DNA formed by renaturation shows approximately 70% recovery of the native structure as judged by its density. With long times of annealing, the DNA can recover the native density. This behavior is also observed with bacterial DNA samples. On the other hand, if precautions arc taken to prevent chain degradation during denaturation, two products appear as a result of renaturation. One of them is undistinguishable from native T4 DNA, whereas the second one consists of highly aggregated DNA which shows only a partial recovery of the native structure. With long times of annealing, this second species recovers the native density but retains its highly aggregated nature. At higher ionic-strengths, renaturation follows a different pattern and a single product is formed. The relevance of all these observations to the kinetic anomalies reported in the previous communication is discussed.     

Renaturation kinetics and thermal stability of DNA in aqueous solutions of formamide and urea.

This paper reports the results of a systematic study of the effects of formamide and urea on the thermal stability and renaturation kinetics of DNA. Increasing concentrations of urea in the range 0 to 8 molar lower the Tm by 2.25 degrees C per molar, and decreases the renaturation rate by approximately 8 percent per molar. Increasing concentrations of formamide in the range from 0 to 50 percent lowers the Tm by 0.60 degrees C per percent formamide for sodium chloride concentrations ranging from 0.035M to 0.88M. At higher salt concentrations the dependence of Tm on percent formamide was found to be slightly greater. Increasing formamide concentration decreases the renaturation rate linearly by 1.1% per percent formamide such that the optimal rate in 50% formamide is 0.45 the optimal rate in an identical solution with no formamide. The effects of urea and formamide on the renaturation rates of DNA are explained by consideration of the viscosities of the solutions at the renaturation temperatures.