Some cytological aspects of antagonism in synthesis of nucleic acid

Summary The effects of solutions of benzimidazole and 5-aminouracil onAllium cepa root meristems immersed in them have been studied cytologically. Benzimidazole and 5-aminouracil, analogues of purines and pyrimidines respectively, reduce the number of nuclear divisions being initiated. A marked decline in frequency of cells in mitosis occurs at the beginning of treatment with benzimidazole (300 p.p.m.) but not until 6 or more hours later with 5-aminouracil (125 p.p.m.).After treatment with 5-aminouracil, small interstitial portions of chromosomes are Feulgen-negative and become extended during anaphase into thin strands. The distal portion of these chromosomes may lag during anaphase and contribute to malformations of the surface of the sister nuclei and to micronuclei-like bodies lying along their adjacent surfaces. However, stickiness resulting in chromosome bridges was not observed. After treatments with benzimidazole, chromosomes are more contracted at metaphase and anaphase than in the control. At the concentrations used, neither component was observed to affect the spindle mechanism of the divisions in progress at the beginning of treatment nor of the divisions which escape inhibition.Reversal of these effects by the introduction of metabolites which these analogues resemble structurally, was investigated. Adenine sulphate (800 p.p.m.) acts antagonistically to benzimidazole (300 p.p.m.) by delaying the marked decline in frequency of mitoses by more than 6 hrs. By 12 hrs. the frequency of cells in mitoses was very low; thus, reversal is not permanent. Half the inhibition of growth caused by 5-aminouracil (50 p.p.m.) is reversed by thymine (50 p.p.m.) for the 24 hrs. of the test period. When 5 p.p.m. folic acid was added to this mixture, root growth exceeded that of the controls. Feulgen-negative gaps, however, are still present in chromosomes at anaphase.Estimations of Feulgen content of nuclei to determine the effect of these treatments on DNA synthesis were made by means of a microphotometer. In control roots there are two classes of nuclei at interphase according to the amount of DNA per nucleus. The amount doubles during interphase before the next division of the nuclei in the root meristem. In roots treated with 5-aminouracil this doubling is inhibited, so that interphase nuclei with the lower amount of DNA accumulate. In roots treated with benzimidazole the relative frequencies of the two classes of nuclei is unaltered; however, mitotic activity is inhibited. This compound apparently affects not only processes concerned with doubling the amount of DNA per nucleus but also processes necessary to the division itself. There was, however, no unquestionable evidence of a nucleus being reconstituted after it had started a division.Contribution from the Program in Cytology, Department of Botany University of Wisconsin, Madison, supported in part by grants to Dr.C. Leonard Huskins from the American Cancer Society, Rockefeller Foundation, and Research Committee of the Graduate School with funds supplied by the Wisconsin Alumni Research Foundation.     

On the fidelity of DNA synthesis. Pyrophosphate-induced misincorporation allows detection of two proofreading mechanisms

The effect of pyrophosphate on the fidelity of in vitro DNA synthesis has been examined. Pyrophosphate enhances misincorporation by Escherichia coli DNA polymerase I in copying phi X174 DNA. The increased misincorporation is directly proportional to the extent of inhibition of the rate of polymerization. In contrast, pyrophosphate is not detectably mutagenic with avian myeloblastosis virus DNA polymerase or DNA polymerases alpha and beta from animal cells, which lack associated proofreading activities. This suggests that increased misincorporation by pyrophosphate is not due to an increase in misinsertions by DNA polymerase, but rather due to inhibition of proofreading by pyrophosphate. However, the pyrophosphate-induced infidelity has a different specificity from, and is not competitive with, two experimental markers of 3'----5' exonuclease proofreading; i.e. the effects of the next nucleotide or the addition of deoxynucleoside monophosphates. These distinctive features suggest a second mode of proofreading susceptible to inhibition by pyrophosphate. This concept is discussed in relation to models for proofreading described in the literature.     

Simulation of interactions in the co-planar nucleic acid base pairs using atom-atom potential functions

Calculations of the energy of nucleic acid base interactions as a function of parameters determining mutual position of two bases in a plane have been performed. Atom-atom potential functions used include terms proportional to the first (electrostatic), sixth (or tenth for the atoms of hydrogen bond) and 12th power of interatomic distance. The calculations have shown the existence of 27 energy minima which correspond to the formation of co-planar pairs with two (or three for G : C pair) almost linear N--H...O and N--H...N hydrogen bonds. The positions of nitrogen bases bound by two hydrogen bonds in every crystal of nucleic acid components, in the complexes of polynucleotides and in tRNA are near to the positions in one of these minima. In addition for every pair there exist energy minima which correspond to the formation of one N--H...O or N--H...N and one C--H...O or C--H...N hydrogen bond. Energy behavior near minima have been investigated. The results of our calculations are in agreement with experimental data and with the calculations which employ quantum mechanical results.     

On the fidelity of deoxyribonucleic acid synthesis directed by chromatin-associated deoxyribonucleic acid polymerase beta

Accuracy of poly[d(A-T)] synthesis catalyzed by chromatin-bound deoxyribonucleic acid (DNA) polymerase beta was measured with several types. A new procedure was developed for the isolation of copied poly[d(A-T)] from chromatin DNA. This method involved in vitro copying of poly[d(A-T)] by native chromatin and subsequent selective fragmentation of chromatin by restriction nucleases, proteinase K, and heat denaturation. The fragmented natural DNA is then separated from the high molecular weight poly[d(A-T)] by gel filtration. The efficacy of DNA removal by this procedure was validated by cesium chloride gradient and nearest-neighbor analysis of the product of the reaction and by measurement of the fidelity of poly[d(A-T)] synthesis by Escherichia coli DNA Pol I contaminated with increasing amounts of DNA. Also, DNA polymerases dissociated from chromatin retain the same accuracy as that of native chromatin. Synthesis of poly[d(A-T)] by chromatin is catalyzed mainly by DNA polymerase-beta. By use of the described technique, we find that the fidelity of this reaction is exceptionally low; approximately one dGTP was incorporated for every thousand complementary nucleotides polymerized.     

Accurate energies of hydrogen bonded nucleic acid base pairs and triplets in tRNA tertiary interactions

Tertiary interactions are crucial in maintaining the tRNA structure and functionality. We used a combined sequence analysis and quantum mechanics approach to calculate accurate energies of the most frequent tRNA tertiary base pairing interactions. Our analysis indicates that six out of the nine classical tertiary interactions are held in place mainly by H-bonds between the bases. In the remaining three cases other effects have to be considered. Tertiary base pairing interaction energies range from -8 to -38 kcal/mol in yeast tRNA(Phe) and are estimated to contribute roughly 25% of the overall tRNA base pairing interaction energy. Six analyzed posttranslational chemical modifications were shown to have minor effect on the geometry of the tertiary interactions. Modifications that introduce a positive charge strongly stabilize the corresponding tertiary interactions. Non-additive effects contribute to the stability of base triplets.     

Pyrophosphate-condensing activity linked to nucleic acid synthesis.

In some preparations of DNA dependent RNA polymerase a new enzymatic activity has been found which catalyzes the condensation of two pyrophosphate molecules, liberated in the process of RNA synthesis, to one molecule of orthophosphate and one molecule of Mg (or Mn) - chelate complex with trimetaphosphate. This activity can also cooperate with DNA-polymerase, on condition that both enzymes originate from the same cells. These results point to two general conclusions. First, energy is conserved in the overall process of nucleic acid synthesis and turnover, so that the process does not require an energy influx from the cell's general resources. Second, the synthesis of nucleic acids is catalyzed by a complex enzyme system which contains at least two separate enzymes, one responsible for nucleic acid polymerization and the other for energy conservation via pyrophosphate condensation.     

Thermodynamical aspects and molecular mechanisms of nerve activity.

This paper proposes a nonequilibrium thermodynamical approach for the energetics of nerve functioning. The energy needs for resting ionic pumping are computed and compared with experimental data on nerve metabolism. A detailed analysis of the various contributions to energetical changes during the nerve impulse clearly shows that without recourse to conformational changes in membrane proteins the initial heat of activity cannot be properly accounted for. By considering the transitions of membrane ionophores, a faithful quantitative explanation of the heat of activity is obtained.     

Theoretical studies on protein-nucleic acid interactions. II. Hydrogen bonding of amino acid side chains with bases and base pairs of nucleic acids

With a view to understanding the role of hydrogen bonds in the recognition of nucleic acids by proteins, hydrogen bonding between the bases and base pairs of nucleic acids and the amino acids (Asn, Gln, Asp and Glu, and charged residues Arg⁺, Glu^?, and Asp^?) has been studied by a second-order perturbation theory. Binding energies have been calculated for all possible configurations involving a pair of hydrogen bonds between the base (or base pair) and the amino acid residue. Our results show that the hydrogen bonding in these cases has a large contribution from electrostatic interaction. In general, the charged amino acids, compared to the uncharged ones, form more stable complexes with bases or base pairs. The hydrogen-bond energies are an order of magnitude smaller than the Coulombic interaction energies between basic amino acids (Lys⁺, Arg⁺, and His⁺) and the phosphate groups of nucleic acids. The stabilities of the complexes of amino acids Asn, Gln, Asp, and Glu with bases are in the order: G–X > C–X > A–X U–X or T–X, and G · C–X > A · T(U)–X, where X is one of these amino acid residues. It has been shown that Glu^? and Asp^? can recognize guanine in single-stranded nucleic acids; Arg⁺ can recognize G · C base pairs from A · T base pairs in double-stranded structures.     

Dampable waves along nucleic acid sequences mediating nucleotides' interactions.

This work studied the relationship of any two nucleotides in genomic sequences, coding sequences and full-length cDNAs. We made a statistical hypothesis that there exist no interactions between any two nucleotides in sequences, therefore, a hypothetical combination distribution of two nucleotides is considered and the difference between the hypothetical combination distribution and the actual distribution is used to measure the average interaction between the two nucleotides. As a result, we found that the interactions between any two nucleotides are clearly and closely related with dampable wavelike patterns along the sequences. Based on the results we daringly make some hypotheses on several biological topics. Further, studies on the wave may provide new clues for gene prediction and genome structure study.     

Experimental studies of molecular interactions between nitrogen bases of nucleic acids.

New experimental results concerning molecular interactions between the nitrogen bases of nucleic acids in the crystalline phase and in vacuo are reported. The temperature dependence of the evaporation rate is measured for solid species. The sensitivity of conventional methods of sublimation heat measurements was improved essentially using a quartz resonator serving as a precise sensor of evaporation rate. Sublimation heats were found for both canonical bases and a number of their derivatives. The in vacuo formation of base associates interacting through hydrogen bonds was observed with a field mass spectrometer. The dimer formation enthalpies, which are indicative of a stronger attraction in complementary pairs compared with noncomplementary ones, were derived from the temperature dependence of ionic currents. Hydrogen-bound complexes of more intricate associates (base trimers and aqueous molecules associates) were studied. The energy gain in the formation of trimers of identical molecules was shown to be larger (per base molecule) than that for dimers.     

Theoretical studies on protein-nucleic acid interactions. III. Stacking of aromatic amino acids with bases and base pairs of nucleic acids

Stacking of aromatic amino acids tryptophan (Trp), tyrosine (Tyr), phenylalanine (Phe), and histidine (His) with bases and base pairs of nucleic acids has been studied. Stacking energies of the amino acid–base (or base pair) complexes have been calculated by second-order perturbation theory. Our results show that, in general, the predominant contribution to the total stacking energy comes from the dispersion terms. In these cases, repulsion energy is greater than the sum of electrostatic and polarization energies. In contrast to this, interaction of histidine with the bases and base pairs is largely Coulombic in nature. The complexes of guanine with aromatic amino acids are more stable than the corresponding complexes of adenine. Among pyrimidines, cytosine forms the most stable complexes with the aromatic amino acids. The G · C base pair has the highest affinity with aromatic amino acids among various sets of base pairs. Optimized geometries of the stacked complexes show that the aromatic moieties overlap only partially. The heteroatom of one residue generally overlaps with the other aromatic moiety. There is a considerable degree of configurational freedom in the stacked geometries. The role of stacking in specific recognition of base sequences by proteins is discussed.     

Reaction mechanisms of riboflavin triplet state with nucleic acid bases.

ESR and laser flash photolysis studies have determined a reasonable order of reactivity of nucleotides with triplet riboflavin (3Rb*) for the first time. ESR detection of triplet state reactivity of Rb with nucleoside, polynucleotide and DNA has been obtained simultaneously. In addition, ESR spin elimination measurement of the reactivity of 3Rb* with nucleotides in good accord with laser flash photolysis determination of the corresponding rate constants offers a simple and reliable method to detect the reactivities of nucleic acids and its components with photoexcited flavins. Kinetic, ESR and thermodynamic studies have demonstrated that Rb should be a strong endogenous photosensitizer capable of oxidizing all nucleic acid bases, and preferentially two purine nucleotides with high rate constants.     

Comments on selected aspects of nucleic acid electrostatics

Recent experimental, theoretical, and computational developments in the field of nucleic acid electrostatics have brought interesting concepts to the fore. The phosphate charge on the double helix apparently influences its structure. When the charge is neutralized asymmetrically, the resulting force imbalance drives bending toward the neutralized side. When the charge is uniformly neutralized, the force imbalance acts to buckle the helical axis, resulting in a compact tertiary conformation. Sharing of condensed counterions by single strands is a stabilizing factor for formation of the double helix. Sharing of condensed counterions by two double helices causes clustering of DNA and may be a factor in RNA folding. Support for these statements is reviewed.