Effects of Escherichia coli SSB protein on the single-stranded DNA-dependent ATPase activity of Escherichia coli RecA protein: Evidence that SSB protein facilitates the binding of RecA protein to regions of secondary structure within single-stranded DNA

The effect that Escherichia coli single-stranded DNA binding (SSB) protein has on the single-stranded DNA-dependent ATPase activity of RecA protein is shown to depend upon a number of variables such as order of addition, magnesium concentration, temperature and the type of single-stranded DNA substrate used. When SSB protein is added to the DNA solution prior to the addition of RecA protein, a significant inhibition of ATPase activity is observed. Also, when SSB protein is added after the formation of a RecA protein-single-stranded DNA complex using either etheno M13 DNA, poly(dA) or poly(dT), or using single-stranded phage M13 DNA at lower temperature (25 °C) and magnesium chloride concentrations of 1 mm or 4 mm, a time-dependent inhibition of activity is observed. These results are consistent with the conclusion that SSB protein displaces the RecA protein from these DNA substrates, as described in the accompanying paper. However, if SSB protein is added last to complexes of RecA protein and single-stranded M13 DNA at elevated temperature (37 °C) and magnesium chloride concentrations of 4 mm or 10 mm, or to poly(dA) and poly(dT) that was renatured in the presence of RecA protein, no inhibition of ATPase activity is observed; in fact, a marked stimulation is observed for single-stranded M13 DNA. A similar effect is observed if the bacteriophage T4-coded gene 32 protein is substituted for SSB protein. The apparent stoichiometry of DNA (nucleotides) to RecA protein at the optimal ATPase activity for etheno M13 DNA, poly(dA) and poly(dT) is 6(±1) nucleotides per RecA protein monomer at 4 mm-MgCl₂ and 37 °C. Under the same conditions, the apparent stoichiometry obtained using single-stranded M13 DNA is 12 nucleotides per RecA protein monomer; however, the stoichiometry changes to 4.5 nucleotides per RecA protein monomer when SSB protein is added last. In addition, a stoichiometry of four nucleotides per RecA protein can be obtained with single-stranded M13 DNA in the absence of SSB protein if the reactions are carried out in 1 mm-MgCl₂. These data are consistent with the interpretation that secondary structure within the natural DNA substrate limits the accessibility of RecA protein to these regions. The role of SSB protein is to eliminate this secondary structure and allow RecA protein to bind to these previously inaccessible regions of the DNA. In addition, our results have disclosed an additional property of the RecA protein-single-stranded DNA complex: namely, in the presence of complementary base-pairing and at elevated temperatures and magnesium concentrations, a unique RecA protein-DNA complex forms that is resistant to inhibition by SSB protein.     

Rad51 protein from the thermotolerant yeast Pichia angusta as a typical but thermodependent member of the Rad51 family

The Rad51 protein from the methylotrophic yeast Pichia angusta (Rad51(Pa)) of the taxonomic complex Hansenula polymorpha is a homolog of the RecA-RadA-Rad51 protein superfamily, which promotes homologous recombination and recombination repair in prokaryotes and eukaryotes. We cloned the RAD51 gene from the cDNA library of the thermotolerant P. angusta strain BKM Y1397. Induction of this gene in a rad51-deficient Saccharomyces cerevisiae strain partially complemented the survival rate after ionizing radiation. Purified Rad51(Pa) protein exhibited properties typical of the superfamily, including the stoichiometry of binding to single-stranded DNA (ssDNA) (one protomer of Rad51(Pa) per 3 nucleotides) and DNA specificity for ssDNA-dependent ATP hydrolysis [poly(dC) > poly(dT) > phiX174 ssDNA > poly(dA) > double-stranded M13 DNA]. An inefficient ATPase and very low cooperativity for ATP interaction position Rad51(Pa) closer to Rad51 than to RecA. Judging by thermoinactivation, Rad51(Pa) alone was 20-fold more thermostable at 37 degrees C than its S. cerevisiae homolog (Rad51(Sc)). Moreover, it maintained ssDNA-dependent ATPase and DNA transferase activities up to 52 to 54 degrees C, whereas Rad51(Sc) was completely inactive at 47 degrees C. A quick nucleation and an efficient final-product formation in the strand exchange reaction promoted by Rad51(Pa) occurred only at temperatures above 42 degrees C. These reaction characteristics suggest that Rad51(Pa) is dependent on high temperatures for activity.     

Purification and characterization of the Thermus thermophilus HB8 RecX protein

The RecA protein plays a central role in homologous recombination by promoting strand exchange between ssDNA and homologous dsDNA. Since RecA alone can advance this reaction in vitro, it is widely used in gene manipulation techniques. The RecX protein downregulates the function of RecA, indicating that it could be used as an inhibitor to control the activities of RecA in vitro. In this study, the RecX protein of the hyper-thermophilic bacterium Thermus thermophilus (ttRecX) was over-expressed in Escherichia coli and purified by heat treatment and several column chromatography steps. Size-exclusion chromatography indicated that purified ttRecX exists as a monomer in solution. Circular dichroism measurements indicated that the alpha-helical content of ttRecX is 54% and that it is stable up to 80 degrees C at neutral pH. In addition, ttRecX inhibited the DNA-dependent ATPase activity of the T. thermophilus RecA protein (ttRecA). The stable ttRecX may be applicable for variety of techniques using the ttRecA reaction.     

Characterization of the helicase activity of the Escherichia coli RecBCD enzyme using a novel helicase assay

We describe an assay to measure the extent of enzymatic unwinding of DNA by a DNA helicase. This assay takes advantage of the quenching of the intrinsic protein fluorescence of Escherichia coli SSB protein upon binding to ssDNA and is used to characterize the DNA unwinding activity of recBCD enzyme. Unwinding in this assay is dependent on the presence of recBCD enzyme and linear dsDNA, is consistent with the known properties of recBCD enzyme, and closely parallels other methods for measuring recBCD enzyme helicase activity. The effects of varying temperature, substrate concentrations, enzyme concentration, and mono- and divalent salt concentrations on the helicase activity of recBCD enzyme were characterized. The apparent Km values for recBCD enzyme helicase activity on linear M13 dsDNA molecules at 25 degrees C are 0.6 nM dsDNA molecules and 130 microM ATP, respectively. The apparent turnover number for unwinding is approximately 15 microM base pairs s-1 (microM recBCD enzyme)-1. When this rate is corrected for the observed stoichiometry of recBCD enzyme binding to dsDNA, kcat for helicase activity corresponds to an unwinding rate of approximately 250 base pairs of DNA s-1 (functional recBCD complex)-1 at 25 degrees C. At 37 degrees C, the apparent Km value for dsDNA molecules was the same as that at 25 degrees C, but the apparent turnover number became 56 microM base pairs s-1 (microM recBCD enzyme)-1 [or 930 base pairs s-1 (functional recBCD complex)-1 when corrected for observed stoichiometry]. With increasing NaCl concentration, kcat peaks at 100 mM, and the apparent Km value for dsDNA increases by 3-fold at 200 mM NaCl. In the presence of 5 mM calcium acetate, the apparent Km value is increased by 3-fold, and kcat decreased by 20-30%. We have also shown that recBCD enzyme molecules are able to catalytically unwind additional dsDNA substrates subsequent to initiation, unwinding, and dissociation from a previous dsDNA molecule.     

fd gene 5 protein binds to double-stranded polydeoxyribonucleotides poly(dA.dT) and poly[d(A-T).d(A-T)]

Circular dichroism (CD) data indicated that fd gene 5 protein (G5P) formed complexes with double-stranded poly(dA.dT) and poly[d(A-T).d(A-T)]. CD spectra of both polymers at wavelengths above 255 nm were altered upon protein binding. These spectral changes differed from those caused by strand separation. In addition, the tyrosyl 228-nm CD band of G5P decreased more than 65% upon binding of the protein to these double-stranded polymers. This reduction was significantly greater than that observed for binding to single-stranded poly(dA), poly(dT), and poly[d(A-T)] but was similar to that observed for binding of the protein to double-stranded RNA [Gray, C.W., Page, G.A., & Gray, D.M. (1984) J. Mol. Biol. 175, 553-559]. The decrease in melting temperature caused by the protein was twice as great for poly[d(A-T).d(A-T)] as for poly(dA.dT) in 5 mM tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), pH 7. Upon heat denaturation of the poly(dA.dT)-G5P complex, CD spectra showed that single-stranded poly(dA) and poly(dT) formed complexes with the protein. The binding of gene 5 protein lowered the melting temperature of poly(dA.dT) by 10 degrees C in 5 mM Tris-HCl, pH 7, but after reducing the binding to the double-stranded form of the polymer by the addition of 0.1 M Na+, the melting temperature was lowered by approximately 30 degrees C. Since increasing the salt concentration decreases the affinity of G5P for the poly(dA) and poly(dT) single strands and increases the stability of the double-stranded polymer, the ability of the gene 5 protein to destabilize poly(dA.dT) appeared to be significantly affected by its binding to the double-stranded form of the polymer.     

Determination of the kinetics of deuteration of DNA.RNA hybrids by ultraviolet spectroscopy

The kinetics of the hydrogen-deuterium exchange reactions of poly(dA).poly(rU) and poly(rA).poly(dT) has been examined, at pH 7.0 and at various temperatures in the 15-35 degrees C range, by stopped-flow ultraviolet spectrophotometry. For comparison, the deuteration kinetics of poly[d(A-T)].poly[d(A-T)] and poly(rA).poly(rU) has been reexamined. At 20 degrees C, the imino deuteration (NH----ND) rates of the two hybrid duplexes were found to be 1.5 and 1.8 s-1, respectively. These are nearly equal to the imino deuteration rates of poly[d(A-T)].poly[d(A-T)] (1.1 s-1) and poly(rA).poly(rU) (1.5 s-1) but appreciably higher than that of poly(dA).poly(dT) (0.35 s-1). It has been suggested that a DNA.RNA hybrid, an RNA duplex, and the AT-alternating DNA duplex have in general higher base-pair-opening reaction rates than the ordinary DNA duplex. The amino deuteration (NH2----ND2) rates, on the other hand, have been found to be 0.25, 0.28, and 0.33 s-1, respectively, for poly(dA).poly(rU), poly(rA).poly(dT), and poly[d(A-T)].poly[d(A-T)], at 20 degrees C. These are appreciably higher than that for poly(rA).poly(rU) (0.10 s-1). In general, the equilibrium constants (K) of the base-pair opening are considered to be greatest for the DNA.RNA hybrid duplex (0.05 at 20 degrees C), second greatest for the RNA duplex (0.02 at 20 degrees C), and smallest for the DNA duplex (0.005 at 20 degrees C), although the AT-alternating DNA duplex has an exceptionally great K (0.07 at 20 degrees C). From the temperature effect on the K value, the enthalpy of the base-pair opening was estimated to be 3.0 kcal/mol for the DNA.RNA hybrid duplex.     

The thermodynamic contribution of the 5-methyl group of thymine in the two- and three-stranded complexes formed by poly(dU) and poly(dT) with poly(dA)

To assess the thermodynamic contribution of the 5-methyl group of thymine, we have studied the two-stranded helical complexes poly(dA).poly(dU) and poly(dA).poly(dT) and the three-stranded complexes--poly(dA).2poly(dU), poly(dA).poly(dT).poly(dU) and poly(dA).2poly(dT)--by differential scanning calorimetry, and uv optical melting experiments. The thermodynamic quantities associated with the 3 --> 2, 2 --> 1, and 3 --> 1 melting transitions are found to vary with salt concentration and temperature in a more complex manner than commonly believed. The transition temperatures, T(m), are generally not linear in the logarithm of concentration or activity of NaCl. The change in enthalpy and in entropy upon melting varies with salt concentration and temperature, and a change in heat capacity accompanies each transition. The poly(dA).2poly(dU) triple helix is markedly different from poly(dA).2poly(dT) in both its CD spectrum and thermodynamic behavior, while the poly(dA).poly(dT).poly(dU) triple helix resembles poly(dA).2poly(dT) in these properties. In comparing poly(dA).2poly(dT) with either the poly(dA).poly(dT).poly(dU) or the poly(dA).2poly(dU) triplexes, the substitution of thymine for uracil in the third strand results in an enhancement of stability against the 3 --> 2 dissociation of deltadeltaG degrees = -135 +/- 85 cal (mol A)(-1) at 37 degrees C. This represents a doubling of the absolute stability toward dissociation compared to the triplexes with poly(dU) as the third strand. The poly (dA).poly (dT) duplex is more stable than poly(dA).poly(dU) by deltadeltaG degrees = -350 +/- 60 cal (mol base pair)(-1) at 37 degrees C. Poly(dA).poly(dT) has 50% greater stability than poly(dA).poly(dU) as a result of the dT for dU substitution in the duplex.     

Changes in the tension in dsDNA alter the conformation of RecA bound to dsDNA�CRecA filaments

The RecA protein is an ATPase that mediates recombination via strand exchange. In strand exchange a single-stranded DNA (ssDNA) bound to RecA binding site I in a RecA/ssDNA filament pairs with one strand of a double-stranded DNA (dsDNA) and forms heteroduplex dsDNA in site I if homology is encountered. Long sequences are exchanged in a dynamic process in which initially unbound dsDNA binds to the leading end of a RecA/ssDNA filament, while heteroduplex dsDNA unbinds from the lagging end via ATP hydrolysis. ATP hydrolysis is required to convert the active RecA conformation, which cannot unbind, to the inactive conformation, which can unbind. If dsDNA extension due to RecA binding increases the dsDNA tension, then RecA unbinding must decrease tension. We show that in the presence of ATP hydrolysis decreases in tension induce decreases in length whereas in the absence of hydrolysis, changes in tension have no systematic effect. These results suggest that decreases in force enhance dissociation by promoting transitions from the active to the inactive RecA conformation. In contrast, increases in tension reduce dissociation. Thus, the changes in tension inherent to strand exchange may couple with ATP hydrolysis to increase the directionality and stringency of strand exchange.     

Temperature dependence of the volumetric parameters of drug binding to poly[d(A-T)].Poly[d(A-T)] and Poly(dA).Poly(dT)

We report the temperature and salt dependence of the volume change (DeltaVb) associated with the binding of ethidium bromide and netropsin with poly(dA).poly(dT) and poly[d(A-T)].poly[d(A-T)]. The DeltaV(b) of binding of ethidium with poly(dA).poly(dT) was much more negative at temperatures approximately 70 degrees C than at 25 degrees C, whereas the difference is much smaller in the case of binding with poly[d(A-T)].poly[d(A-T)]. We also determined the volume change of DNA-drug interaction by comparing the volume change of melting of DNA duplex and DNA-drug complex. The DNA-drug complexes display helix-coil transition temperatures (Tm several degrees above those of the unbound polymers, e.g., the Tm of the netropsin complex with poly(dA)poly(dT) is 106 degrees C. The results for the binding of ethidium with poly[d(A-T)].poly[d(A-T)] were accurately described by scaled particle theory. However, this analysis did not yield results consistent with our data for ethidium binding with poly(dA).poly(dT). We hypothesize that heat-induced changes in conformation and hydration of this polymer are responsible for this behavior. The volumetric properties of poly(dA).poly(dT) become similar to those of poly[d(A-T)].poly[d(A-T)] at higher temperatures.     

A 5'----3' exoribonuclease of Saccharomyces cerevisiae: size and novel substrate specificity

The purification scheme for a 5'----3' exoribonuclease of Saccharomyces cerevisiae has been modified to facilitate purification of larger amounts of enzyme and further extended to yield highly purified enzyme by use of poly(A)-agarose chromatography. As determined by either sodium dodecyl sulfate-polyacrylamide gel electrophoresis or physical characterization, the enzyme has a molecular weight of about 160,000. Further studies of its substrate specificity show that poly(C) and poly(U) preparations require 5' phosphorylation for activity and that poly(A) with a 5'-triphosphate end group is hydrolyzed at only 12% of the rate of poly(A) with a 5'-monophosphate end group. DNA is not hydrolyzed, but synthetic polydeoxyribonucleotides are strong competitive inhibitors of the hydrolysis of noncomplementary ribopolymers. Poly(A).poly(U) and poly(A).poly(dT) are hydrolyzed at 60 and 50%, respectively, of the rate of poly(A) at 37 degrees C. The RNase H activity of the enzyme can also be demonstrated using an RNA X M13 DNA hybrid as a substrate. When poly(dT).poly(dA) with a 5'-terminal poly(A) segment on the poly(dA) is used as a substrate, the enzyme hydrolyzes the poly(A) "tail," removing the last ribonucleotide, but does not hydrolyze the poly(dA).     

A homopolymer of the potent antitumor drug 2'-deoxy-5-fluorouridylate

Poly(5-fluoro-2'-deoxyuridylic acid) was synthesized and its properties were compared with those of poly(dT) and poly(dU). It readily complexed with poly(dA). The 1:1 complex melted at about 20 degrees C lower than poly(dA) . poly(dT). A triple-stranded helix, poly(dA) . 2 poly(dF5U) was formed only in high salt (2.0 M NaCl).     

Novel properties of DNA polymerase beta with poly(rA).oligo(dT) template-primer.

Purified DNA polymerase beta of calf thymus can utilize poly(rA).oligo(dT) as efficiently as poly(dA).oligo(dT) or activated DNA as a template primer. The poly(rA).oligo(dT)-dependent activity of DNA polymerase beta was found to differ markedly from the DNA-dependent activity of the same enzyme (with either activated calf thymus DNA or poly(dA).(dT)10) in the following respects. 1) Poly(rA)-dependent activity was strongly inhibited by natural DNA from various sources or synthetic deoxypolymer duplexes at very low concentrations (less than 0.5 microgram/ml) at which the DNA-dependent activity was affected to a much smaller extent, if at all. 2) Poly(rA)-dependent activity was inhibited by N-ethylmaleimide more strongly than DNA-dependent activity measured at 37 degrees C, while it was resistant to this reagent at 26 degrees C. 3) The curves of the activity versus substrate concentration were sigmoidal in the poly(rA)-dependent reaction but hyperbolic in the activated DNA-dependent reaction. A kinetic study suggested that the association of beta-enzyme protomers may be required to copy the poly(rA) strand.     

Effect of the strandedness of cofactor DNA on the activities of DNA-dependent ATPases B and C3 from FM3A cells

The requirements of cofactor DNA for DNA-dependent ATPases B and C3 were analyzed in detail. ATPase B and C3 required the presence of a polynucleotide for their activities. Among the DNAs tested, ATPase B showed a preference for poly(dT) as its cofactor. The other deoxyhomopolymers, except poly(dG) and heat-denatured DNA also were effective. The alternating polydeoxyribonucleotide, poly[d(A-T)] had an efficiency 23% that of heat-denatured DNA. Unlike ATPase B, ATPase C3 showed almost no activity with deoxyhomopolymers. The most effective cofactor for ATPase C3 so far tested is poly[d(A-T)]. Relatively high activity was obtained with heat-denatured DNA. The high activity of ATPase B with poly(dT) was reduced by the addition of poly(dA). The addition of noncomplementary homopolymers did not affect enzyme activity. ATPase C3 activity in the presence of 10 microM poly(dT) increased gradually with concentrations of poly(dA) up to 20 microM, after which it decreased. Almost no increase in activity was observed when noncomplementary homopolymers were added. The relatively high activity of ATPase C3 with heat-denatured DNA was suggested by its high sensitivity to ethidium bromide to be due to the double-stranded region in the heat-denatured DNA formed by self-annealing.     

Interaction of the HMG1 protein with nucleic acids

Binding constants have been measured for the interaction of the protein HMG1 with native DNA, denatured DNA and a number of polynucleotides at near-physiological ionic strengths, using gel filtration and thermal denaturation. The interaction of HMG1 with DNA is shown to be noncooperative and reversible. Nucleic acids form the following series in order of increasing binding constants: poly(U) integral of poly(A) less than poly(dA) less than dsDNA integral of poly(dA) X poly(dT) integral of poly(dG) X poly(dC) much less than poly[d(A-T]) integral of ssDNA.     

DNA sequence dependence of ATP hydrolysis by RecA protein

The DNA sequence dependence of the ATPase activity of RecA protein has been investigated for a variety of single strand octamer and hexadecamer homopolymers and alternating copolymers. Under assay conditions where the single strand DNA concentration exceeds the RecA protein concentration, significant differences in the rates of ATP hydrolysis for the various single strand DNA oligomer cofactors are observed. Under the conditions examined, the order of efficiency of the DNA cofactors in inducing RecA mediated ATPase activity is found to be: dA16 greater than dT16 greater than d(TC)16 greater than dT8 greater than dC16 greater than dA8 = dG8 greater than dG16 greater than dC8 greater than d(AG)16. These results demonstrate not only a dependence of RecA ATPase activity on the sequence composition of short single strand DNA they further reveal ATPase activity can be affected by the nearest neighbor nucleotide sequence of short DNA cofactors.     

Multiple deoxyribonucleic acid dependent adenosinetriphosphatases in FM3A cells. Characterization of an adenosinetriphosphatase that prefers poly [d(A-T)] as cofactor

Four chromatographically distinct DNA-dependent ATPases, B, C1, C2, and C3, have been partially purified from mouse FM3A cell extracts. These ATPases are distinguished from each other by their physical and enzymological properties. DNA-dependent ATPases B, C1, C2, and C3 have sedimentation coefficients in 250 mM KCl of 5.5, 5.3, 7.3, and 3.4 S, respectively. ATPases B, C2, and C3 hydrolyze dATP as efficiently as ATP, whereas C1 does not. ATPase B hydrolyzes other ribonucleoside triphosphates with relatively high efficiency as compared to the other three enzymes. ATPase C3 prefers poly[d(A-T)] to poly(dT) as cofactor, whereas the other three enzymes prefer poly(dT) to poly[d(A-T)]. Among the four ATPases, ATPase C3 has been highly purified and characterized in detail. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the most purified fraction of ATPase C3 showed two major bands corresponding to molecular weights of 66 000 and 63 000. The Km values of the enzyme for ATP and dATP are 0.53 and 0.86 mM, respectively. As cofactor, poly[d(A-T)] is the most effective among the DNAs tested. Heat-denatured DNA and native DNA are also effective but used with less efficiency. Almost no or very little activity has been detected with ribohomopolymers and oligonucleotides. The activity attained with poly(dT) and poly(dA) is 11 and 6% of that with heat-denatured DNA, respectively. When both polymers were added at a molar ratio 1 to 1, very high activity was obtained with these polymers. On the other hand, little activity was observed by the combination of noncomplementary homopolymers such as poly(dT) and poly(dG).     

Physical studies of DNA premelting equilibria in duplexes with and without homo dA.dT tracts: correlations with DNA bending

We have employed a variety of physical methods to study the equilibrium melting and temperature-dependent conformational dynamics of dA.dT tracts in fractionated synthetic DNA polymers and in well-defined fragments of kinetoplast DNA (kDNA). Using circular dichroism (CD), we have detected a temperature-dependent, "premelting" event in poly(dA).poly(dT) which exhibits a midpoint near 37 degrees C. Significantly, we also detect this CD "premelting" behavior in a fragment of kDNA. By contrast, we do not observe this "premelting" behavior in the temperature-dependent CD spectra of poly[d(AT)].poly[d(AT)], poly(dG).poly(dC), poly[d(GC)].poly[d(GC)], or calf thymus DNA. Thus, poly(dA).poly(dT) and kDNA exhibit a common CD-detected "premelting" event which is absent in the other duplex systems studied in this work. Furthermore, we find that the anomalous electrophoretic retardation of the kDNA fragments we have investigated disappears at temperatures above approximately 37 degrees C. We also observe that the rotational dynamics of poly(dA).poly(dT) and kDNA as assessed by singlet depletion anisotropy decay (SDAD) and electric birefringence decay (EBD) also display a discontinuity near 37 degrees C, which is not observed for the other duplex systems studied. Thus, in the aggregate, our static and dynamic measurements suggest that the homo dA.dT sequence element [common to both poly(dA).poly(dT) and kDNA] is capable of a temperature-dependent equilibrium between at least two helical states in a temperature range well below that required to induce global melting of the host duplex. We suggest that this "preglobal" melting event may correspond to the thermally induced "disruption" of "bent" DNA.     

Specificity and kinetics defining the interaction between a murine monoclonal autoantibody and DNA

Interactions between a murine monoclonal anti-DNA autoantibody (BV17-45) and DNA were examined by direct binding and competitive radioimmunoassays. Binding isotherms constructed by titration of purified BV17-45 with a series of distinct 32P-labeled double-stranded DNA ([32P]dsDNA) fragments were super-impossible, suggesting: 1) BV17-45/[32P]dsDNA binding is independent of dsDNA size using fragments greater than or equal to 192 base pairs in length, and 2) BV17-45 does not exhibit stringent sequence specificity. Single-stranded DNA-specific monoclonal antibody BV04-01 did not react with [32P]dsDNA, confirming its duplex character. In competition experiments, BV17-45 cross-reacted with phage (phi X174, M13) RF AND VIRION DNAS AT PICOMOLAR concentrations. Selectivity for B-form DNA was suggested by the ability of poly(dA) . poly(dT), but not other helical duplex forms, to block BV17-45/[32P] dsDNA binding. Among the four deoxyribohomopolymers, only deoxyadenylic acid polymers completely inhibited BV17-45/[32P]dsDNA complex formation. [32P]dsDNA binding was relatively insensitive to ionic strength, suggesting minimal contribution of electrostatic forces to the binding free energy. Measured BV17-45/[32P]dsDNA association and dissociation rate constants (4 degrees C) were 7.4 X 10(6) M-1 s-1 and 9.2 X 10(-5) s-1, respectively, yielding a functional affinity of 8 X 10(10) M-1. Results are discussed in terms of the relative contribution of B-DNA structural and substructural determinants to the mechanism of BV17-45 recognition.     

The Mcm467 complex of Saccharomyces cerevisiae is preferentially activated by autonomously replicating DNA sequences

We have analyzed the role of single-stranded DNA (ssDNA) in the modulation of the ATPase activity of Mcm467 helicase of the yeast Saccharomyces cerevisiae. The ATPase activity of the Mcm467 complex is modulated in a sequence-specific manner and that the ssDNA sequences derived from the origin of DNA replication of S. cerevisiae autonomously replicating sequence 1 (ARS1) are the most effective stimulators. Synthetic oligonucleotides, such as oligo(dA) and oligo(dT), also stimulated the ATPase activity of the Mcm467 complex, where oligo(dT) was more effective than oligo(dA). However, the preference of a thymidine stretch appeared unimportant, because with yeast ARS1 derived sequences, the A-rich strand was as effective in stimulating the ATPase activity, as was the T-rich strand. Both of these strands were more effective stimulators than either oligo(dA)( )()or oligo(dT). The DNA helicase activity of Mcm467 complex is also significantly stimulated by the ARS1-derived sequences. These results indicate that the ssDNA sequences containing A and B1 motifs of ARS1, activate the Mcm467 complex and stimulate its ATPase and DNA helicase activities. Our results also indicate that the yeast replication protein A stimulated the ATPase activity of the Mcm467 complex.     

Microcalorimetric investigation of DNA, poly(dA)poly(dT) and poly[d(A-C)]poly[d(G-T)] melting in the presence of water soluble (meso tetra (4 N oxyethylpyridyl) porphyrin) and its Zn complex

Thermodynamic parameters of melting process (DeltaHm, Tm, DeltaTm) of calf thymus DNA, poly(dA)poly(dT) and poly(d(A-C)).poly(d(G-T)) were determined in the presence of various concentrations of TOEPyP(4) and its Zn complex. The investigated porphyrins caused serious stabilization of calf thymus DNA and poly poly(dA)poly(dT), but not poly(d(A-C))poly(d(G-T)). It was shown that TOEpyp(4) revealed GC specificity, it increased Tm of satellite fraction by 24 degrees C, but ZnTOEpyp(4), on the contrary, predominantly bound with AT-rich sites and increased DNA main stage Tm by 18 degrees C, and Tm of poly(dA)poly(dT) increased by 40 degrees C, in comparison with the same polymers without porphyrin. ZnTOEpyp(4) binds with DNA and poly(dA)poly(dT) in two modes--strong and weak ones. In the range of r from 0.005 to 0.08 both modes were fulfilled, and in the range of r from 0.165 to 0.25 only one mode--strong binding--took place. The weak binding is characterized with shifting of Tm by some grades, and for the strong binding Tm shifts by approximately 30-40 degrees C. Invariability of DeltaHm of DNA and poly(dA)poly(dT), and sharp increase of Tm in the range of r from 0.08 to 0.25 for thymus DNA and 0.01-0.2 for poly(dA)poly(dT) we interpret as entropic character of these complexes melting. It was suggested that this entropic character of melting is connected with forcing out of H2O molecules from AT sites by ZnTOEpyp(4) and with formation of outside stacking at the sites of binding. Four-fold decrease of calf thymus DNA melting range width DeltaTm caused by increase of added ZnTOEpyp(4) concentration is explained by rapprochement of AT and GC pairs thermal stability, and it is in agreement with a well-known dependence, according to which DeltaT approximately TGC-TAT for DNA obtained from higher organisms (L. V. Berestetskaya, M. D. Frank-Kamenetskii, and Yu. S. Lazurkin. Biopolymers 13, 193-205 (1974)). Poly (d(A-C))poly(d(G-T)) in the presence of ZnTOEpyp(4) gives only one mode of weak binding. The conclusion is that binding of ZnTOEpyp(4) with DNA depends on its nucleotide sequence.