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Branched DNA molecules arise transiently as intermediates in genetic recombination or on extrusion of cruciforms from covalent circular DNA duplexes that contain palindromic sequences. The free energy of these structures relative to normal DNA duplexes is of interest both physically and biologically. Oligonucleotide complexes that can form stable branched structures, DNA junctions, have made it possible to model normally unstable branched states of DNA such as Holliday recombinational intermediates. We present here an evaluation of the free energy of creating four-arm branch points in duplex DNA, using a system of two complementary junctions and four DNA duplexes formed from different combinations of the same set of eight 16-mer strands. The thermodynamics of formation of each branched structure from the matching pair of intact duplexes have been estimated in two experiments. In the first, labeled strands are allowed to partition between duplexes and junctions in a competition assay on polyacrylamide gels. In the second, the heats of forming branched or linear molecules from the component strands have been determined by titration microcalorimetry at several temperatures. Taken together these measurements allow us to determine the standard thermodynamic parameters for the process of creating a branch in an otherwise normal DNA duplex. The free energy for reacting two 16-mer duplexes to yield a four-arm junction in which the branch site is incapable of migrating is + 1.1 (+/- 0.4) kcal mol-1 (at 18 degrees C, 10 mM-Mg2+). Analysis of the distribution of duplex and tetramer products by electrophoresis confirms that the free energy difference between the four duplexes and two junctions is small at this temperature. The associated enthalpy change at 18 degrees C is +27.1 (+/- 1.3) kcal mol-1, while the entropy is +89 (+/- 30) cal K-1 mol-1. The free energy for branching is temperature dependent, with a large unfavorable enthalpy change compensated by a favorable entropy term. Since forming one four-stranded complex from two duplexes should be an entropically unfavorable process, branch formation is likely to be accompanied by significant changes in hydration and ion binding. A significant apparent delta Cp is also observed for the formation of one mole of junction, +0.97 (+/-0.05) kcal deg-1 mol-1.
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PMID:Thermodynamics of DNA branching. 154 18

Twelve monoclonal antibodies against cobrotoxin from Naja naja atra venom were tested for cross-reactivity with eight different snake toxins, binding to linear epitopes, prevention of cobrotoxin binding to acetylcholine receptor (AchR) in vitro, and protection in mice concomitantly given a lethal dose of cobrotoxin. The antibodies were highly specific, as evidenced by little reactivity with other snake toxins. None of the monoclonal antibodies bound to reduced cobrotoxin or synthesized 8-mer regions spanning the whole molecule, thus suggesting the recognition of conformational epitopes. The in vitro binding of toxin to AchR was competitively inhibited (23-79%) with a 1.66:1 mole ratio of antibody:AchR. Preincubation of monoclonal antibody with toxin before adding AchR (3:1 mole ratio of AchR:antibody) inhibited the in vitro binding of toxin to AchR by 20-80%. Monoclonal antibodies added after the preincubation of toxin with AchR did not dissociate the toxin-AchR complex. An antibody:toxin mole ratio of 2.5:1, with 6 micrograms of cobrotoxin, delayed the time to death of mice 3.7-23.8-fold over control mice. The monoclonal antibodies that most effectively prevented in vitro binding of toxin to AchR also provided the longest delay in time to death in mice.
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PMID:Production and characterization of monoclonal antibodies against Naja naja atra cobrotoxin. 172 6

HMG 14 and protamine can be used to enhance intermolecular ligation of low concentrations of linear DNA. Adding HMG 14 (50 moles per mole DNA) caused 50% of blunt-ended DNA to form predominantly dimers, and all cohesive-ended DNA to form multimers (greater than 6-mer) in response to T4 ligase. Protamine was maximally effective at 40:1, producing mostly dimers and trimers. Adding higher concentrations of HMG 14 did not affect the ligation pattern of cohesive-ended DNA, while higher concentrations of protamine inhibit the formation of multimers. Phosphorylation of HMG 14 at Ser 20 by Ca(++)-phospholipid dependent protein kinase abolished the ability of HMG 14 to stimulate intermolecular ligation, but did not substantially interfere with intramolecular ligation, or the binding of HMG 14 to linear or circular DNA as assessed by gel mobility. Thus Ser 20, which is located in the amino terminal DNA-binding domain of HMG 14, appears to modulate DNA-DNA interactions.
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PMID:HMG 14 and protamine enhance ligation of linear DNA to form linear multimers: phosphorylation of HMG 14 at Ser 20 specifically inhibits intermolecular DNA ligation. 184 50

The transcription factor GAL4 from Saccharomyces cerevisiae contains a "zinc-finger"-like motif, Cys-Xaa2-Cys-Xaa6-Cys-Xaa6-Cys-Xaa2-Cys-Xaa6+ ++-Cys, within its DNA-binding domain. A GAL4 fragment consisting of residues 1-147 plus two additional residues from the cloning vector [denoted GAL4(149*)] has been cloned and overexpressed in Escherichia coli. This fragment includes the entire DNA-binding domain (residues 1-74). The homogeneous GAL4-(149*) protein contains 1-1.5 moles of Zn(II) per mole of protein. The GAL4(149*) protein binds tightly to the specific 17-base-pair palindromic DNA sequence found at GAL4 binding sites as shown by gel-retention assays using a 32P-labeled 23-mer containing this sequence. Removal of the intrinsic Zn(II) by EDTA at low pH abolishes binding to the 23-mer. The GAL4(149*) apoprotein can be reconstituted with Zn(II), Cd(II), or Co(II) with restoration of specific DNA binding. Titration of GAL4(149*) apoprotein with 113Cd(II) shows two 113Cd(II) binding sites on the molecule, one with delta of 707 ppm, suggesting coordination to four sulfur atoms, and one with delta of 669 ppm, suggesting coordination to three or four sulfur atoms. Because GAL4(149*) protein contains only six cysteine residues within its DNA-binding domain, the precise coordination of the two Cd(II) ions cannot be stated with certainty; one or more shared -S- ligands could exist. GAL4(149*) protein contains approximately 40% alpha-helix and approximately 20% beta-sheet, estimated from circular dichroism. Removal of the native Zn(II) ion causes limited unfolding of secondary structure, but less than one turn of alpha-helix. The binding of Zn(II), Cd(II), and, to a lesser extent, Co(II) to GAL4(149*) apoprotein protects the protein from proteolysis by trypsin, which produces a 13-kDa DNA-binding core.
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PMID:Structure and function of the Zn(II) binding site within the DNA-binding domain of the GAL4 transcription factor. 249 63

HCV helicase [E(wt)] catalyzed strand separation of a short DNA duplex (F21:HF31) formed from a 5'-hexachlorofluorescein-tagged 31-mer (HF31) and a 3'-fluorescein-tagged 21-mer (F21) complementary to the 5'-end of HF31. Strand separation was monitored by the fluorescence increase associated with the formation of F21 from F21:HF31. In the presence of ATP, the strand-separating activity was catalytic. In the absence of ATP and with E(wt) concentrations greater than that of F21:HF31, a biphasic fluorescence increase was observed at 25 degrees C. The late phase of this reaction was assigned to the separation of F21 from F21:HF31. The ATP-independent strand-separating reaction occurred more rapidly in the absence of Mg(2+) than in its presence. This result correlated with a lower T(m) value of F21:HF31 in the absence of 3.5 mM Mg(2+) than in its presence (45 vs 63 degrees C). The stoichiometry for the strand-separating reaction in the absence of ATP was 8 mol of E(wt) per mole of F21:HF31 separated into single-stranded F21 and HF31. The dissociation constants of HCV helicase for F21, HF31, and F21:HF31 in the absence of Mg(2+) were 0.6 +/- 0.4, 6 +/- 1, and 7.3 +/- 0.9 nM, respectively. Histidinyl-tagged E(wt) [hE(wt)] and a mutant enzyme [hE(V432A)] were prepared. hE(wt) and E(wt) bound F21 and HF31 with similar affinities and had similar ATP-dependent helicase activities, whereas hE(V432A) bound F21 and HF31 with affinities similar to that of E(wt) but had greatly reduced ATP-dependent helicase activities. In contrast to E(wt) and hE(wt), hE(V432A) did not support the ATP-independent strand-separating reaction. Consequently, the ATP-independent strand-separating reaction was not only the result of the high affinity of the enzyme for single-stranded DNA. The enzyme preferentially used duplex DNA with a 3'-tail for the ATP-dependent helicase reaction. In contrast, the enzyme strand-separated blunt-ended, 5'-tailed, and 3'-tailed duplex DNA equally effectively in the ATP-independent strand-separating reaction.
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PMID:Strand-separating activity of hepatitis C virus helicase in the absence of ATP. 1081 84

Despite extensive investigations into oligonucleotide lipoplexes, virtually no work has addressed whether the physicochemical properties of these assemblies vary as a function of the constituent oligonucleotide (ODN) sequence and/or composition. The present study was aimed at answering this question. To this end, we complexed N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP) liposomes, in dispersion, with either 18-mer phosphorothiote homo-oligonucleotides composed of either adenine, thymidine or cytosine; or one of three structurally related 18-mer phosphorothioate oligonucleotides (S-ODNs) (G3139, its reverse sequence and its two-base mismatch). After ODN addition to vesicles at different mole ratios, changes in pH and electrical surface potential at the lipid-water interface were analyzed by using the fluorophore heptadecyl-7-hydroxycoumarin while particle size distributions were analyzed by static-light scattering. The results indicate that each homo-oligonucleotide does indeed exhibit different complexation behavior. In particular, the maximal level of DOTAP neutralization by the polyadenine S-ODN is much lower than that for the two other homo-oligonucleotides and hence its lipoplex is much more positively charged. Much smaller electrostatic differences are also apparent between lipoplexes formed from each of the G3139-related ODNs. This paper identifies nucleotide base selection and sequence as a variable that can affect the physicochemical properties of oligonucleotide lipoplexes and hence probably their transfection competency.
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PMID:Oligonucleotide lipoplexes: the influence of oligonucleotide composition on complexation. 1178 23

Polypyridyl complexes of Co decorated with 350-Da polyether chains (Co(350)(2+)) form molten phases of nucleic acids when paired with DNA counterions (Co(350)DNA) or 25-mer oligonucleotides. Analysis of voltammetry and chronoamperometry of mixtures of these phases with complexes having ClO(4)(-) counterions (Co(350)(ClO(4))(2)) and no other diluent provides charge transport rates from the oxidation and reduction currents for the complexes. As the mole fraction of the Co(350)(ClO(4))(2) complex in the mixture is varied from ca. 0.25 to 1, the physical diffusion constants derived from the Co(III/II) wave increase from 1 x 10(-11) cm(2)/s to 5 x 10(-10) cm(2)/s, and apparent diffusion constants dominated by the Co(II/I) electron self-exchange increase from 1 x 10(-10) cm(2)/s to 2 x 10(-8) cm(2)/s. Pure Co(350)DNA melts, containing no Co(350)(ClO(4))(2) complex, do not exhibit recognizable voltammetric waves; DNA suppresses the Co(II/I) electron transfer reactions of Co complexes for which it is the counterion. There are therefore two microscopically distinct kinds of Co(350) complexes, those with DNA and those with ClO(4)(-) counterions, with respect to their Co(II/I) electron-transfer dynamics, leading to percolative behavior in their mixtures. The electron-transfer rates of the Co(II/I) couple are controlled by the diffusive relaxation of the ionic atmosphere around the reaction pair, and the inactivity of the bound Co complexes can be attributed to the very low mobility of the anionic phosphate groups in the DNA counterion. Substitution of sulfonated polystyrene for DNA produced similar results, suggesting that this phenomenon is general to other polymer counterions of low mobility. We conclude that the measured Co(II/I) charge transport and electron-transfer rate constants reflect more the diffusive mobility of the perchlorate counterion than the intrinsic Co(II/I) electron hopping rate.
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PMID:Ion atmosphere relaxation and percolative electron transfer in Co bipyridine DNA molten salts. 1276 89

The total metabolic cost of soybean (Glycine max L. Mer Clark) nodule nitrogen fixation was empirically separated into respiration associated with electron flow through nitrogenase and respiration associated with maintenance of nodule function.Rates of CO(2) evolution and H(2) evolution from intact, nodulated root systems under Ar:O(2) atmospheres decreased in parallel when plants were maintained in an extended dark period. While H(2) evolution approached zero after 36 hours of darkness at 22 degrees C, CO(2) evolution rate remained at 38 degrees of the rate measured in light. Of the remaining CO(2) evolution, 62% was estimated to originate from the nodules and represents a measure of nodule maintenance respiration. The nodule maintenance requirement was temperature dependent and was estimated at 79 and 137 micromoles CO(2) (per gram dry weight nodule) per hour at 22 degrees C and 30 degrees C, respectively.The cost of N(2) fixation in terms of CO(2) evolved per electron pair utilized by nitrogenase was estimated from the slope of H(2) evolution rate versus CO(2) evolution rate. The cost was 2 moles CO(2) evolved per mole H(2) evolved and was independent of temperature.In this symbiosis, nodule maintenance consumed 22% of total respiratory energy while the functioning of nitrogenase consumed a further 52%. The remaining respiratory energy was calculated to be associated with ammonia assimilation, transport of reduced N, and H(2) evolution.
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PMID:Experimental determination of the respiration associated with soybean/rhizobium nitrogenase function, nodule maintenance, and total nodule nitrogen fixation. 1666 99

The effects of potassium ion on the nested allostery of GroEL are due to increases in the affinity for nucleotide. Both positive allosteric transitions, TT-TR and TR-RR, occur at lower [ATP] as [K(+)] is increased. Negative cooperativity in the double-ringed system is also due to an increase in the affinity of the trans ring for the product ADP as [K(+)] is increased. Consequently, (i) rates of ATP hydrolysis are inversely proportional to [K(+)] and (ii) the residence time of GroES bound to the cis ring is prolonged and the hemicycle time extended. Substrate protein suppresses negative cooperativity by decreasing the affinity of the trans ring for ADP, reducing the hemicycle time to a constant minimum. The trans ring thus serves as a variable timer. ATP added to the asymmetric GroEL-GroES resting-state complex lacking trans ring ADP is hydrolyzed in the newly formed cis ring with a presteady-state burst of approximately 6 mol of Pi per mole of 14-mer. No burst is observed when the trans ring contains ADP. The amplitude and kinetics of ATP hydrolysis in the cis ring are independent of the presence or absence of encapsulated substrate protein and independent of K(+) at concentrations where there are profound effects on the linear steady-state rate. The hydrolysis of ATP by the cis ring constitutes a second, nonvariable timer of the chaperonin cycle.
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PMID:Setting the chaperonin timer: the effects of K+ and substrate protein on ATP hydrolysis. 1898 45

The human bodily defense system includes a wide variety of innate antimicrobial proteins. Histatins are small molecular weight proteins produced by the human salivary glands that exhibit antifungal and antibacterial activities. While evolutionarily old salivary proteins such as mucins and proline-rich proteins contain large regions of tandem repeats, relatively young proteins like histatins do not contain such repeated domains. Anticipating that domain duplications have a functional advantage, we genetically engineered variants of histatin 3 with one, two, three, or four copies of the functional domain by PCR and splice overlap. The resulting proteins, designated reHst3 1-mer, reHist3 2-mer, reHis3 3-mer and reHist3 4-mer, exhibited molecular weights of 4,062, 5,919, 7,777, and 9,634 Da, respectively. The biological activities of these constructs were evaluated in fungicidal assays toward Candida albicans blastoconidia and germinated cells. The antifungal activities per mole of protein increased concomitantly with the number of functional domains present. This increase, however, was higher than could be anticipated from the molar concentration of functional domains present in the constructs. The demonstrated increase in antifungal activity may provide an evolutionary explanation why such domain multiplication is a frequent event in human salivary proteins.
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PMID:Anti-candidal activity of genetically engineered histatin variants with multiple functional domains. 2325 51


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