Purification and characterization of the coliphage N4-coded single-stranded DNA binding protein

We have purified and characterized a single-stranded DNA binding protein (N4 SSB) induced after coliphage N4 infection. It has a monomeric molecular weight of 31,000 and contains 10 tyrosine and 1-2 tryptophan amino acid residues. Its fluorescence spectrum is dominated by the tyrosine residues, and their fluorescence is quenched when the protein binds single-stranded DNA. Fluorescence quenching was used as an assay to quantitate binding of the protein to single-stranded nucleotides. The N4 single-stranded DNA binding protein binds cooperatively to single-stranded nucleic acids and binds single-stranded DNA more tightly than RNA. The binding involves displacement of cations from the DNA and anions from the protein. The apparent binding affinity is very salt-dependent, decreasing as much as 1,000-fold for a 10-fold increase in NaCl concentration. The degree of cooperativity (omega) is relatively independent of salt concentration. At 37 degrees C in 0.22 M NaCl, the protein has an intrinsic binding constant for M13 viral DNA of 3.8 x 10(4) M-1, a cooperativity factor omega of 300, and binding site size of 11 nucleotides per monomer. The protein lowers the melting point of poly(dA.dT).poly(dA-dT) by greater than 60 degrees C but cannot lower the melting transition or assist in the renaturation of natural DNA. N4 single-stranded DNA binding protein enhances the rate of DNA synthesis catalyzed by the N4 DNA polymerase by increasing the processivity of the N4 DNA polymerase and melting out hairpin structures that block polymerization.     

Purification and properties of yolk protein factor I, a sequence-specific DNA-binding protein from Drosophila melanogaster

We report the purification and some of the biochemical properties of yolk protein factor I (YPF1). This protein binds to a specific site in the yolk protein 1 gene (yp1) of Drosophila melanogaster. YPF1 has been purified to 95% homogeneity and consists of a heterodimer of two subunits with molecular weights 85,000 and 69,000. The protein is highly asymmetric with a frictional ratio of 1.56 which leads to calculated dimensions of 510 x 51 A when modeled as a prolate ellipsoid of revolution. It binds the yp1 DNA site with a protein/DNA stoichiometry of 1:1. Binding to that site is essentially irreversible with a dissociation rate constant of koff less than or equal to 2 x 10(-7) s-1, which gives the complex a dissociation half-life of approximately 55 days. The measured apparent second order association rate constant is 4 x 10(8) M-1 s-1 resulting in a calculated equilibrium dissociation constant of KD less than or equal to 5 x 10(-16) M. YPF1 also has a 10(8) selectivity for the yp1 site over poly(dA).poly(dT) (KDapp = 2 x 10(-8) M(nucleotide].     

Characterization of RNA aptamer binding by the Wilms' tumor suppressor protein WT1

The interaction of the zinc finger protein WT1 with RNA aptamers has been investigated using a quantitative binding assay, and the results have been compared to those from a previous study of the DNA binding properties of this protein. A recombinant peptide containing the four zinc fingers of WT1 (WT1-ZFP) binds to representatives of three specific families of RNA aptamers with apparent dissociation constants ranging from 13.8 +/- 1.1 to 87.4 +/- 10.4 nM, somewhat higher than the dissociation constant of 4.12 +/- 0.4 nM for binding to DNA. An isoform that contains an insertion of three amino acids between the third and fourth zinc fingers (WT1[+KTS]-ZFP) also binds to these RNAs with slightly reduced affinity (the apparent dissociation constants ranging from 22.8 to 69.8 nM) but does not bind to DNA. The equilibrium binding of WT1-ZFP to the highest-affinity RNA molecule was compared to the equilibrium binding to a consensus DNA molecule as a function of temperature, pH, monovalent salt concentration, and divalent salt concentration. The interaction of WT1-ZFP with both nucleic acids is an entropy-driven process. Binding of WT1-ZFP to RNA has a pH optimum that is narrower than that observed for binding to DNA. Binding of WT1-ZFP to DNA is optimal at 5 mM MgCl(2), while the highest affinity for RNA was observed in the absence of MgCl(2). Binding of WT1 to both nucleic acid ligands is sensitive to increasing monovalent salt concentration, with a greater effect observed for DNA than for RNA. Point mutations in the zinc fingers associated with Denys-Drash syndrome have dramatically different effects on the interaction of WT1-ZFP with DNA, but a consistent and modest effect on the interaction with RNA. The role of RNA sequence and secondary structure in the binding of WT1-ZFP was probed by site-directed mutagenesis. Results indicate that a hairpin loop is a critical structural feature required for protein binding, and that some consensus nucleotides can be substituted provided proper base pairing of the stem of the hairpin loop is maintained.     

Escherichia coli thioredoxin stabilizes complexes of bacteriophage T7 DNA polymerase and primed templates

The DNA polymerase activity induced after bacteriophage T7 infection of Escherichia coli is found in a complex of two proteins, the T7 gene 5 protein and a host protein, thioredoxin. Gene 5 protein is a DNA polymerase and a 3' to 5' exonuclease. Thioredoxin binds tightly to the gene 5 protein and increases the processivity of polymerization some 1000-fold. Gene 5 protein forms a short-lived complex with the primer-template, poly(dA).oligo(dT), in the absence of Mg2+ and nucleotides. Thioredoxin increases the half-life of the preformed primer-template-polymerase complex from less than a second to approximately 5 min. The dissociation is accelerated by excess single-stranded DNA in an apparent second order reaction, indicating direct transfer of polymerase between DNA fragments. Thioredoxin also reduces the equilibrium dissociation constant, Kd, of the gene 5 protein -poly(dA).oligo(dT) complex 20- to 80-fold. The salt dependence of Kd indicates that thioredoxin stabilizes the primer-template-polymerase complex mainly through additional charge-charge interactions, increasing the estimated number of interactions from 2 to 7. The affinity of gene 5 protein for single-stranded DNA is at least 1000-fold higher than for double-stranded DNA and is little affected by thioredoxin. Under conditions of steady state synthesis the effect of thioredoxin on the polymerization rate is determined by two competing factors, an increase in processivity and a decrease of the dissociation rate of polymerase and replicated template.     

Investigation of binding between recA protein and single-stranded polynucleotides with the aid of a fluorescent deoxyribonucleic acid derivative

The availability of epsilon DNA, a fluorescent ssDNA derivative, has made it possible to examine quantitatively the interactions between recA protein and single-stranded polynucleotides. Fluorescence titrations of epsilon DNA with recA protein and vice versa establish that each recA protein monomer covers 5.5 epsilon DNA nucleotides and that the dissociation constant of the recA-epsilon DNA complex is 10 nM. Fluorescence titrations of recA protein-epsilon DNA mixtures with poly(dT) establish that each recA protein monomer covers 5.1 poly(dT) nucleotides and that the dissociation constant of the recA-poly(dT) complex is 0.03 nM. Observations on how the addition of ssDNA affects the fluorescence of recA protein-epsilon DNA mixtures establish that the dissociation constant of the recA-ssDNA complex exceeds 20 microM. Stopped-flow kinetics in which excess recA protein binds to epsilon DNA indicate that k2 = 6 X 10(6) M-1 s-1 for the process. A more approximate kinetic technique indicates that recA protein binds to epsilon DNA at least one-tenth as fast as to poly(dT); the rate constant for dissociation of recA-epsilon DNA exceeds that for recA-poly(dT) by at least 30-fold. epsilon DNA is proven to be a versatile reagent for studying single-stranded polynucleotide-protein interactions. Not only can its own complexes with protein be investigated but also, under suitable circumstances, it can be used as a fluorescent probe to explore complexes incorporating nonfluorescent polynucleotides.     

Mammalian heterogeneous nuclear ribonucleoprotein A1. Nucleic acid binding properties of the COOH-terminal domain

A1 is a core protein of the eukaryotic heterogeneous nuclear ribonucleoprotein complex and is under study here as a prototype single-stranded nucleic acid-binding protein. A1 is a two-domain protein, NH2-terminal and COOH-terminal, with highly conserved primary structure among vertebrate homologues sequenced to date. It is well documented that the NH2-terminal domain has single-stranded DNA and RNA binding activity. We prepared a proteolytic fragment of rat A1 representing the COOH-terminal one-third of the intact protein, the region previously termed COOH-terminal domain. This purified fragment of 133 amino acids binds to DNA and also binds tightly to the fluorescent reporter poly(ethenoadenylate), which is used to access binding parameters. In solution with 0.41 M NaCl, the equilibrium constant is similar to that observed with A1 itself, and binding is cooperative. The purified COOH-terminal fragment can be photochemically cross-linked to bound nucleic acid, confirming that COOH-terminal fragment residues are in close contact with the polynucleotide lattice. These binding results with isolated COOH-terminal fragment indicate that the COOH-terminal domain in intact A1 can contribute directly to binding properties. Contact between both COOH-terminal domain and NH2-terminal domain residues in an intact A1:poly(8-azidoadenylate) complex was confirmed by photochemical cross-linking.     

Tryptic core protein of lactose repressor binds operator DNA.

The core protein produced by mild proteolytic digestion of lactose repressor protein has been purified from native repressor by chromatography on phosphocellulose. The core protein isolated in this manner binds to operator DNA with an apparent dissociation constant of 10(-7) M, and the observed binding is decreased by the presence of inducer. Competition studies with nonspecific DNA indicate that the binding species in the core protein preparations is neither intact lactose repressor nor mixed tetramers containing varying numbers of intact NH2-terminal regions. This conclusion is supported by experiments designed to measure the rate of dissociation of the core protein from the operator DNA. Calculations based on the assumption that the isolated core protein binds similarly to the corresponding region in intact repressor protein indicate that the core region contributes approximately 40 to 50% of the energy of binding to operator DNA. Furthermore, the change in operator affinity upon inducer binding to core accounts for a minimum of 60% of the free energy change in binding to operator observed for the native protein. The demonstration that core protein binds to operator DNA requires a re-evaluation of the various models for repressor binding to DNA. A possible model based on the available information is presented.     

Interaction of phloretin with the anion transport protein of the red blood cell membrane

Phloretin is an inhibitor of anion exchange and glucose and urea transport in human red cells. Equilibrium binding and kinetic studies indicate that phloretin binds to band 3, a major integral protein of the red cell membrane. Equilibrium phloretin binding has been found to be competitive with the binding of the anion transport inhibitor, 4,4′-dibenzamido-2,2′-disulfonic stilbene (DBDS), which binds specifically to band 3. The apparent binding (dissociation) constant of phloretin to red cell ghost band 3 in 28.5 mM citrate buffer, pH 7.4, 25°C, determined from equilibrium binding competition, is $\text{1.8 ± 0.1}\text{μM}$. Stopped-flow kinetic studies show that phloretin decreases the rate of DBDS binding to band 3 in a purely competitive manner, with an apparent phloretin inhibition constant of $\text{1.6 ± 0.4}\text{μM}$. The pH dependence of equilibrium binding studies show that it is the charged, anionic form of phloretin that competes with DBDS binding, with an apparent phloretin inhibition constant of 1.4 μM. The phloretin binding and inhibition constants determined by equilibrium binding, kinetic and pH studies are all similar to the inhibition constant of phloretin for anion exchange. These studies suggest that phloretin inhibits anion exchange in red cells by a specific interaction between phloretin and band 3.     

Comparative kinetics of cofactor association and dissociation for the human and trypanosomal S-adenosylhomocysteine hydrolases. 1. Basic features of the association and dissociation processes

The S-adenosyl-l-homocysteine (AdoHcy) hydrolases catalyze the reversible conversion of AdoHcy to adenosine and homocysteine, making use of a catalytic cycle in which a tightly bound NAD+ oxidizes the 3-hydroxyl group of the substrate at the beginning of the cycle, activating the 4-CH bond for elimination of homocysteine, followed by Michael addition of water to the resulting intermediate and a final reduction by the tightly bound NADH to give adenosine. The equilibrium and kinetic properties of the association and dissociation of the cofactor NAD+ from the enzymes of Homo sapiens (Hs-SAHH) and Trypanosoma cruzi (Tc-SAHH) are qualitatively similar but quantitatively distinct. Both enzymes bind NAD+ in a complex scheme. The four active sites of the homotetrameric apoenzyme appear to divide into two numerically equal classes of active sites. One class of sites binds cofactor weakly and generates full activity very rapidly (in less than 1 min). The other class binds cofactor more strongly but generates activity only slowly (>30 min). In the case of Tc-SAHH, the final affinity for NAD+ is roughly micromolar and this affinity persists as the equilibrium affinity. In the case of Hs-SAHH, the slow-binding phase terminates in micromolar affinity also, but over a period of hours, the dissociation rate constant decreases until the final equilibrium affinity is in the nanomolar range. The slow binding of NAD+ by both enzymes exhibits saturation kinetics with respect to the cofactor concentration; however, binding to Hs-SAHH has a maximum rate constant around 0.06 s-1, while the rate constant for binding to Tc-SAHH levels out at 0.006 s-1. In contrast to the complex kinetics of association, both enzymes undergo dissociation of NAD+ from all four sites in a single first-order reaction. The equilibrium affinities of both Hs-SAHH and Tc-SAHH for NADH are in the nanomolar range. The dissociation rate constants and the slow-binding association rate constants for NAD+ show a complex temperature dependence with both enzymes; however, the cofactor always dissociates more rapidly from Tc-SAHH than from Hs-SAHH, the ratio being around 80-fold at 37 degrees C, and the cofactor binds more rapidly to Hs-SAHH than to Tc-SAHH above approximately 16 degrees C. These features present an opening for selective inhibition of Tc-SAHH over Hs-SAHH, demonstrated with the thioamide analogues of NAD+ and NADH. Both analogues bind to Hs-SAHH with approximately 40 nM affinities but much more weakly to Tc-SAHH (0.6-15 microM). Nevertheless, both analogues inactivated Tc-SAHH 60% (NAD+ analogue) or 100% (NADH analogue) within 30 min, while the degree of inhibition of Hs-SAHH approached 30% only after 12 h. The rate of loss of activity is equal to the rate of dissociation of the cofactor and thus 80-fold faster at 37 degrees C for Tc-SAHH.     

Effects of the Escherichia coli SSB protein on the binding of Escherichia coli RecA protein to single-stranded DNA. Demonstration of competitive binding and the lack of a specific protein-protein interaction

The effect of the Escherichia coli single-stranded DNA binding (SSB) protein on the stability of complexes of E. coli RecA protein with single-stranded DNA has been investigated through direct DNA binding experiments. The effect of each protein on the binding of the other to single-stranded DNA, and the effect of SSB protein on the transfer rate of RecA protein from one single-stranded DNA molecule to another, were studied. The binding of SSB protein and RecA protein to single-stranded phage M13 DNA is found to be competitive and, therefore, mutually exclusive. In the absence of a nucleotide cofactor, SSB protein binds more tightly to single-stranded DNA than does RecA protein, whereas in the presence of ATP-gamma-S, RecA protein binds more tightly than SSB protein. In the presence of ATP, an intermediate result is obtained that depends on the type of DNA used, the temperature, and the magnesium ion concentration. When complexes of RecA protein, SSB protein and single-stranded M13 DNA are formed under conditions of slight molar excess of single-stranded DNA, no effect of RecA protein on the equilibrium stability of the SSB protein-single-stranded DNA complex is observed. Under similar conditions, SSB protein has no observed effect on the stability of the RecA protein-etheno M13 DNA complex. Finally, measurements of the rate of RecA protein transfer from RecA protein-single-stranded DNA complexes to competing single-stranded DNA show that there is no kinetic stabilization of the RecA protein-etheno M13 DNA complex by SSB protein, but that a tenfold stabilization is observed when single-stranded M13 DNA is used to form the complex. However, this apparent stabilizing effect of SSB protein can be mimicked by pre-incubation of the RecA protein-single-stranded M13 DNA complex in low magnesium ion concentration, suggesting that this effect of SSB protein is indirect and is mediated through changes in the secondary structure of the DNA. Since no direct effect of SSB protein is observed on either the equilibrium or dissociation properties of the RecA protein-single-stranded DNA complex, it is concluded that the likely effect of SSB protein in the strand assimilation reaction is on a slow step in the association of RecA protein with single-stranded DNA. Direct evidence for this conclusion is presented in the accompanying paper.     

Analysis of DNA-RecA protein interactions involving the protein self-association reaction

A method to analyze the DNA binding properties of RecA protein is developed to take into account the protein self-association reaction and is applied to reanalyze the interaction with chemically modified single-stranded DNA (epsilon-DNA). Protein oligomerization is investigated by static light-scattering measurements and analyzed with the accumulated strain model. A coupled equilibrium between DNA binding and self-association of RecA is resolved considering that the complex formed between an m polymer (where m represents the number of units of the polymer) and DNA is identical to the complex resulting from the cooperative binding of m monomers. The cooperativity parameter thus determined is about 10(4), which is more than 100 times higher than the apparent parameter estimated without consideration of the protein oligomerization. This extremely high figure is in good agreement with the formation of large clusters of complex observed by electron microscopy. The apparent DNA binding constant depends upon the ratio of the DNA binding affinity and the self-association constant. For this reason, the variation of the DNA binding constant with the salt concentration is amplified, and the number of ion pairs formed between DNA and RecA obtained from the apparent salt dependence (11 ion pairs/monomer) has been overestimated. Only 2 ion pairs may be formed.     

Intercalators as probes of DNA conformation: propidium binding to alternating and non-alternating polymers containing guanine

Propidium iodide is used as a structural probe for alternating and non-alternating DNA polymers containing guanine and the results are compared to experiments with poly[d(A-T)2], poly(dA . dT) and random DNA sequences. Viscometric titrations indicate that propidium binds to all polymers and to DNA by intercalation. The binding constant and binding site size are quite similar for all alternating polymers, non-alternating polymers containing guanine and natural DNA. Poly(dA . dT) is unusual with a lower binding constant and positive cooperativity in its propidium binding isotherms. Poly(dA . dT) and poly(dG . dC) have similar salt effects but quite different temperature effects in propidium binding equilibria. Polymers and natural DNA have similar rate constants in their SDS driven dissociation reactions. The association rate constants are similar for the alternating polymers and poly(dG . dC) but are significantly reduced for poly(dA . dT). These results suggest that natural DNA, the alternating polymers, and non-alternating polymers containing guanine convert to an intercalated conformation with bound propidium in a very similar manner.     

Evolutionary conservation and DNA binding properties of the Ssh7 proteins from Sulfolobus shibatae

The thermoacidophilic archaeon Sulfolobus shibatae synthesizes a large amount of the 7-ku DMA binding proteins known as Ssh7. Our hybridization experiments showed that two Ssh7-encoding genes existed in the genome of S. shibatae. These two genes, designated ssh7a and ssh7b, have been cloned, sequenced and expressed in Escherichia coli. The two Ssh7 proteins differ only at three amino acid positions. In addition, the cis-regulatory sequences of the ssh7a and ssh7b genes are highly conserved. These results suggest the presence of a selective pressure to maintain not only the sequence but also the expression of the two genes. We have also found that there are two genes encoding the 7-ku protein in Sulfolobus solfataricus. Based on this and other studies, we suggest that the gene encoding the 7-ku protein underwent duplication before the separation of Sulfolobus species. Binding of native Ssh7 and recombinant (r)Ssh7 to short duplex DNA fragments was analyzed by electrophoretic mobility shift assays. Both n     

Purification of the cytochalasin B binding component of the human erythrocyte monosaccharide transport system.

The cytochalasin B binding component of the human erythrocyte monosaccharide transport system has been purified. The preparation appears to contain one major protein with an apparent polypeptide chain molecular weight of 55,000 and about 0.4 binding sites per chain. Cytochalasin B binds to the reconstituted preparation with a dissociation constant of 1.3.10(-7) M, a value which is similar to that reported for the transport system in the intact erythrocyte.     

Cryospectrokinetic evidence for the mode of reversible binding of neocarzinostatin chromophore to poly(deoxyadenylic-thymidylic acid)

The spectra of neocarzinostatin (NCS) chromophore during its reversible association with poly(dA-dT).poly(dA-dT) [poly(dA-dT)] were recorded (at intervals of 17 ms or more) by a cryospectroscopic method. Examination of the spectral changes of a drug during its interaction with DNA has not been previously reported. Such studies indicate binding of chromophore to poly(dA-dT) is a two-step process in which the spectral properties of the intermediate poly(dA-dT). NCS chromophore species closely resemble those of the final equilibrium species. On the basis of cryokinetic studies (at single wavelengths) carried out at low temperature (2 degrees C), the following proposed mechanism of the DNA-drug (PD) interaction was quantitated: (Formula: see text). In analogy with the other reports on the kinetics of drug-DNA interaction, (PD)I and (PD)II could represent externally bound and intercalated complexes, respectively. However, since the spectra of (PD)I and (PD)II are closely similar, it can also be proposed that (PD)I and (PD)II represent two forms of an intercalated complex. The rate and equilibrium constant for each step were determined by examining the kinetics of the forward and reverse reactions. This was accomplished by determining the polynucleotide concentration dependence of the apparent fast and slow first-order rate constants observed during a double-exponential increase in transmittance (at 330 nm) associated with the binding and the apoprotein-induced dissociation rate constant of the chromophore from poly(dA-dT). The opportunity to use apoprotein, instead of a detergent, to follow the kinetics of the reverse reaction provides a novel approach to these studies.(ABSTRACT TRUNCATED AT 250 WORDS)     

DNA-binding properties of gene-5 protein encoded by bacteriophage M 13. 1. The kinetics of the dissociation of gene-5-protein.polynucleotide complexes upon addition of salt

The irreversible dissociation kinetics of complexes of M13-encoded gene-5 protein with the polynucleotides poly(dA) and M13 DNA was studied by means of stopped-flow experiments. A linear decay was found for all gene-5-protein.poly(dA) complexes and for the gene-5-protein.M13 DNA complexes for which the DNA lattice was completely saturated at the beginning of the dissociation experiments. Only at the end of the dissociation curve was a deviation from linearity observed. A single-exponential decay was found for the dissociation of gene-5-protein.M13 DNA complexes when the DNA was not completely saturated initially. These results could be interpreted by assuming that dissociation of bound protein is only possible from isolated binding sites, while during the dissociation, rearrangement of bound protein clusters takes place continuously, including the formation of newly isolated bound protein. This redistribution results from a translocation of the protein along the lattice, which, for the poly(dA) complex, is fast with respect to the dissociation step, but which is slow for the M13 DNA complex. During this process the equilibrium cluster distribution predicted by the theory of McGhee and Von Hippel is not maintained. The binding of gene-5 protein to poly(dA) or poly(dT) does not result in a broadening of the nucleotide resonances in the NMR spectra of these polynucleotides, as had been observed for E. coli DNA-binding protein and interpreted as an indication for a high rate of translocation of the protein on the polynucleotide. The absence of line broadening for gene-5-protein.polynucleotide complexes is caused by the high binding cooperativity. As a consequence the majority of the protein molecules are bound in a cluster which makes the concentration of isolated bound protein very low. This results in a decrease of the signal/noise ratio at higher degrees of binding, but does not lead to line broadening while fast translocation still occurs.     

Oxidized dimeric Scapharca inaequivalvis. Co-driven perturbation of the redox equilibrium

The dimeric hemoglobin isolated from Scapharca inaequivalvis, HbI, is notable for its highly cooperative oxygen binding and for the unusual proximity of its heme groups. We now report that the oxidized protein, an equilibrium mixture of a dimeric high spin aquomet form and a monomeric low spin hemichrome, binds ferrocyanide tightly which allows for internal electron transfer with the heme iron. Surprisingly, when ferricyanide-oxidized HbI is exposed to CO, its spectrum shifts to that of the ferrous CO derivative. Gasometric removal of CO leads to the oxidized species rather than to ferrous deoxy-HbI. At equilibrium, CO binds with an apparent affinity (p50) of about 10-25 mm of Hg and no cooperativity (20 degrees C, 10-50 mM buffers at pH 6.1). The kinetics of CO binding under pseudo-first order conditions are biphasic (t1/2 of 15-50 s at pH 6.1). The rates depend on protein, but not on CO concentration. The nitrite-oxidized protein is not reduced readily in the presence of CO unless one equivalent of ferrocyanide, but not of ferricyanide, is added. We infer that ferrocyanide, produced in the oxidation reaction, is tightly bound to the protein forming a redox couple with the heme iron. CO shifts the redox equilibrium by acting as a trap for the reduced heme. The equilibrium and kinetic aspects of the process have been accounted for in a reaction scheme where the internal electron transfer reaction is the rate-limiting step.