The effect of bases non-complementary to the template on the efficacy of primer interaction with the Klenow fragment of DNA polymerase I from Escherichia coli

The comparison of the Km and Vmax values for the primers was carried out. The primers were either completely complementary to the template or contained non-complementary bases at different positions with respect to the 3'-end. The addition of NaF, selectively inhibiting 3'----5'-exonuclease activity of the enzyme, was shown to result in the increase of Vmax values by 10% and 30% for complementary and partially complementary primers, respectively, Km values of the latters being unchanged. Km values for d[(pT)10pC] is about 146-fold greater than that for d[(pT)11]. Km values for d[(pT)7pC(pT)2] (20 microM) and d[[(pT)2pC]3pT] (20 microM); d[(pT)4pC(pT)5] (5.0 microM); d[(pC)(pT)7] (1.3 microM) and d[(pT)2pC(pT)7] (1.2 microM) are comparable with those for d[(pT)2] (22 microM), d[(pT)5] (4.1 microM) and d[(pT)7] (1.2 microM), respectively, but not with the decathymidylate d[(pT)10] (0.2 microM). We suggest that it is not the length of the primers but the number of bases in the fragment beginning with the first nucleotide from the 3'-end and ending in the non-complementary base, that determines the efficiency of interaction of the primers containing non-complementary bases with the enzyme. The addition of one link to d(pT)n (n less than or equal to 10) resulted in a 1.8-fold increase in the affinity. When 11 less than n less than 25 the affinity is decreased so that d(pT)22-23 have minimal affinity to the enzyme. The primers containing more than 50 units were found to have about the same affinity (calculated on base concentration) as d(pT)10-11.     

The effect of bases noncomplementary to the template on the effectiveness of interaction between primers and avian myeloblastosis virus reverse transcriptase]

The comparison of the Km and vmax values for various primers was carried out. The primers were either completely complementary to the poly(A)-template or contained noncomplementary bases in different positions from the 3'-end. An increase of the Km and vmax values for primers containing noncomplementary bases was shown. The affinity of the AMV-revertase complex with poly(A)-template to d(pT)10 was shown to be higher by a factor of 93, 325, 338, 425, 95 and 15 than to d(pT)9(pC), d[(pT)2pC]3pT, d(pT)8pCpT, d(pT)7pC(pT)2, d(pT)4pC(pT)5 and d(pC)3(pT)7, respectively. The vmax values for the above primers were 1.2-1.5-fold higher than for d(pT)10. The decrease of the affinity of noncorrect primers to the enzyme was supposed to serve as a mechanism for mistakes correction when noncomplementary to the template mononucleotide units were added to the primer. More effective discrimination between right and wrong primers takes place if the noncomplementary base is in the second or third position from the 3'-end. The mistake correction is performed by dissociation of a wrong primer from the complexes with the enzyme and template. The data obtained for AMV-revertase are in accord with results for pro- and eukaryotic DNA polymerases and are in favour of a similar mechanism of mistake correction by all enzymes in the case of short primers.     

Comparison of the effectiveness of the interaction of ribo- and deoxyriboprimers with DNA-polymerase from the human placenta

The Km and Vmax values for primers d(pA)n, d(pT)n, r(pA)n, r(pU)n where n = 1-16, were compared. The Km values for minimal primers dTMP, dAMP, rUMP, rAMP were found to be 48, 71, 602 and 602 microM, respectively. The Vmax value for any NMP made up approximately 7% of that for (pN)10. The lengthening of any primer per one mononucleotide unit for n from 1 to 10 resulted in the decrease of the Km value 1.8-fold and the increase of the Vmax value 1.35-fold. The ratios of the Km values for primers r(pA)n-d(pA)n and r(pU)n-d(pT)n were 7.5 and 12.5, respectively, for any n. The Km value for [d[pT)8]r(pU) primer was the same as for r(pU)9, but not for d(pT)9. Decanucleotide [d(Tp)9]ddT interacted with the polymerase competitively to the template, but not to the primer. The primer's 3'-OH group was supposed to form the hydrogen bond with the enzyme. The absence of 3'-hydroxygroup in [d(Tp)9]ddT resulted in its inability to compete effectively with the primer. The difference of the affinity of ribo- and deoxyriboprimers is due, apparently, to the existence of the different conformation of the furanose rings in the ribose and deoxyribose.     

Comparison of initiating abilities of primers of different length in polymerization reactions catalyzed by DNA polymerases from thermoacidophilic archaebacteria

Optimal conditions for polymerization reaction catalyzed on poly(dA) and poly(dT) templates by DNA polymerases from thermoacidophilic archaebacteria--DNA polymerase A from Sulfolobus acidocaldarius and DNA polymerase B from Thermoplasma acidophilum--have been established. Values of Km and Vmax (60 degrees C) for a set of primers d(pA)n and d(pT)n have been estimated. Minimal primers for both enzymes are dNMP. Lengthening of primers by each mononucleotide increases their affinity about 2.16-fold. Linear dependence of log Km and of log vmax on the number of mononucleotide links in primers (n) has breaking point at n = 10. The value of Vmax is about 20% of that for decanucleotide. The affinity of the primer d(pA)9p(rib*) with a deoxyribosylurea residue at the 3'-end does not differ essentially from that of d(pA)9. Substitution of the 3'-terminal nucleotide of a complementary primer for a noncomplementary nucleotide, e.g., substitution of 3'-terminal A for C in d(pA)10 in the reaction catalyzed on poly(dT), decreases the affinity of a primer by one order of magnitude.     

The algorithm of estimation of the Km values for primers of various structure and length in the polymerization reaction catalyzed by Klenow fragment of DNA polymerase I from E. coli

DNA synthesis at primers d(pT)n, d(pA)n, d(pC)n, and d(pG)n in the presence of corresponding complementary templates and at hetero-oligoprimers complementary to M13 phage DNA was investigated. The values of both -log Km and log Vmax increased linearly if homo-oligoprimers contained less than 10 nucleotides. The lengthening of d(pT)n and d(pA)n primers by one mononucleotide unit (n = 1-10) resulted in the 1.82-fold decrease of the Km values. The incremental decreases of Km for d(pC)n and d(pG)n were equal to about 2.46. The enhancement of the homo- and hetero-oligonucleotide primers' affinity to the enzyme due to one Watson-Crick hydrogen bond between complementary template and primer is about 1.35 times. This allows to calculate the Km values for primers of various structure and length up to 10 units. The objective laws of the Km and Vmax values changes for primers containing more than 10 nucleotides were analyzed.     

E. coli DNA polymerase. A study of the mechanism of primer binding using oligothymidylate analogs with ethylated internucleotide phosphate groups

The following individual diastereomers of oligothymidylate ethyl esters (the alkyl phosphodiester group is asymmetric with R or S configuration) have been prepared: d[(Tr)8Tp'(Et)T] (I), d[(Tp)8Tp'(Et)T] (II), d[(Tp)8Tp'(Et)TpT] (III), d[(Tp)8Tp' X (Et)TpT] (IV). A totally esterified analogue d[[(Tp(Et)7]T] (V) was obtained as a diastereomeric mixture. All oligothymidylate derivatives revealed substrate activity as primers of DNA polymerase with poly(dA) as a template. The values of the maximal reaction rates were equal to 14; 2,6; 68; 24 and 0,1% for oligothymidylates (I)-(V) with respect to Vm value (100%) for (Tp)9T. Km values of oligothymidylates (I)-(V), 2,7; 2,5; 0,51; 7,2 microM, were obtained in relation to Km for d[(Tp)9T] (0,4 microM). Diastereomers (I) and (II) were not destroyed by Klenow fragment of DNA polymerase I which has only 3'----5' exonuclease activity. However, these derivatives were hydrolyzed by complete DNA polymerase I due to its 5'----3' exonuclease activity, the reaction rate being 3-10 times lower than in case of d[(Tp)9T]. The data suggest an essential contribution to the primer binding from the positive enzyme group interaction with the 3'-end negatively charged phosphate group of oligonucleotide, together with the primer complementary interaction with the template. At least two phosphodiester groups of the oligonucleotide primer are essential for the reaction of polymerization following the correct binding.     

DNA-polymerase alpha from human placenta. Effectiveness of interaction between oligothymidylates of different lengths and the template-binding site

Modification of human placenta DNA polymerase alpha by (pT)2pC[Pt2 + (NH3)2OH].(pT)7 was investigated. The linear time dependence of the enzyme activity logarithm suggested a pseudo-first order for modification. Kd value of enzyme-affinity reagent complex (0.5 microM) was estimated. The enzyme inactivation by the affinity reagent and protection from inactivation in the presence of oligonucleotides of varying length were used for determining Kd values of the enzyme-ligand complexes. Oligonucleotide d(pT)2pC(pT)7 (Kd 0.15 microM), d(Tp)9T (Kd 0.15 microM) and [d(Tp)9]ddT (Kd 0.15 microM) protected the enzyme from inactivation with equal efficiency. The protective action of oligothymidylates d(Tp)nT (where n changes from 3 to 14) strongly depended on the chain length, the Kd values diminishing from 5.3 to 0.0091 microM in the geometrical progression. The addition of one link to the oligothymidylate chain resulted in 1.71-fold increase in the oligonucleotide affinity for the enzyme specific site. Such a change corresponds to Gibbs energy change of about 0.32 kcal/mole. It is supposed that the monomer units of pentadecathymidylate (at least beginning with the third one) in d(Tp)14T-enzyme complex form neither hydrogen bonds nor electrostatic linkages with the enzyme. Kd values of oligonucleotides as templates are shown to reflect quite well the true affinity of template for the enzyme. This affinity increases in the presence of a primer. However, the ratio of the affinity for different oligonucleotides does not change in the presence or absence of a complementary primer.     

5'-derivatives of oligonucleotides as primers of DNA polymerization catalyzed by AMV reverse transcriptase and Klenow fragment of DNA polymerase 1.

The Km and Vmax values for d(pT)8 and its derivatives containing various 5'-end groups were estimated in the reaction of polymerization catalyzed with AMV-RT and FK. The change in affinity of modified primers was more pronounced in the case of AMV-RT than in the case of FK. Introducing in d(pT)8 of intercalators such as phenazinium, ethidium and daunomycin residues results in 2.7-, 8.7- and 11-fold increases in the primer affinity to AMV-RT, respectively. However, in the case of hemin and cholesterol derivatives the Km values were 3 and 5 times higher than those for d(pT)8. Compared to d(pT)8, the affinity of FK to all the above analogs was 2.3-3.6 times higher with the exception of cholesterol derivative to which it was 2.4-fold lower. The effect of the 5'-end residues on the Vmax values of d(pT)8 was small and ranged from 44% to 120% of that for d(pT)8. Therefore such reactive derivatives of oligonucleotides can be used as effective primers of AMV-RT and FK. Possible reasons for various effects of the 5'-end residues of the primer on its interaction with FK or AMV-RT in the presence of poly(A) are discussed.     

Dependence of 3'-5-exonuclease activity of a fragment of Klenow DNA polymerase I from Escherichia coli on the length and structure of the cleaved oligonucleotide

The Km and vmax values for oligothymidylates d(pT)2-16 in reaction of 3'-5'-exonuclease hydrolysis catalyzed by Klenow fragment were measured in the absence and presence of poly(dA) template without the poly(dA), the Km values for oligonucleotides are slightly dependent on their length. The rate of oligothymidylates hydrolysis increases with their length and for d(pT)16 it is about 190-times higher than for d(pT)2. The addition on poly(dA) does not lead to an essential change of the Km values for d(pT)2-16, but raises the rate of d(pT)2-7 hydrolysis 2-17-fold and at the same time lowers the efficiency of d(pT)8-16 hydrolysis. The Km values for d(pC)10, d(pA)19 and d(pT)10 are nearly the same. However the velocity of d(pC)10 hydrolysis is approximately 1,2 and 7,8-times higher than for d(pA)10 and d(pC)10, respectively d(pC)10, d(pA)10 and d(pT)10 under conditions of interaction with the template-binding site raise the rate of hydrolysis of d(pT)2 combined with the exonuclease center, with various efficiency. Under similar conditions, d(pT)8, d(pT)10 and d(pT)16 as templates activated hydrolysis of d(pT)2. The dependence of the Klenow fragment exonuclease activity both on the length and structure of the template and on the length of the hydrolyzed oligonucleotide was suggested.     

Structure-function analysis of mononucleotides and short oligonucleotides in the priming of enzymatic DNA synthesis

The reversed-phase chromatography technique was employed in the measurement of DNA synthesis at the primers d(pT)n, r(pU)n, d(pA)n, and r(pA)n (n = 1-16) in the presence of template poly(dA) or poly(dT). DNA synthesis was catalyzed by Escherichia coli DNA polymerase I Klenow fragment, Physarum polycephalum DNA polymerase beta-like, P. polycephalum DNA polymerase alpha, and human placenta DNA polymerase alpha. Values of Km and Vmax were measured as functions of the primer chain lengths. It was found that all mononucleotides and small oligonucleotides served as primers of DNA synthesis. Values of the logarithm of both Km and Vmax increased linearly until primers had attained a chain length of 9-12 nucleotides, where a break was observed. The incremental as well as the absolute values of Km were interpreted in terms of free binding energies. These together with other data indicate that the 3'-ultimate nucleotide of the primer contributes a decisive amount of free energy of binding to DNA polymerase both from the nucleoside and from the phosphate moiety. The incremental increase is due to a complementary interaction between bases of primer and template buried in the binding cleft of the polymerase. It is also the ultimate nucleotide that determines whether the ribonucleotide or the deoxyribonucleotide is an efficient primer. It is of interest that the major results seem preserved for all four DNA polymerases. An energetic model for the binding of the template-primer was proposed and compared with available crystallographic data.     

Klenow fragment of DNA-polymerase I from E. coli. III. The role of internucleotide phosphate groups of the matrix in its binding with the enzyme

The modification of Klenow fragment of DNA polymerase I E. coli was investigated by the affinity reagents d(Tp)2C[Pt2+(NH3)2OH](pT)7 and d(pT)2pC[Pt2+(NH3)2OH](pT)7. The template binding site of the enzyme was modified by these reagents in the presence of NaF (5 mM), which inhibits selectively the 3'----5'-exonuclease activity of the enzyme and therefore prevents the reagent from degradation. NaCN destroyed covalent bonds between reagents and enzyme, restoring activity of the Klenow fragment. The affinity of different ligands (inorganic phosphate, nucleoside monophosphates, oligonucleotides) to the template binding site of Klenow fragment was estimated. Minimal ligands capable to bind with the template site were shown to be triethylphosphate (Kd 290 microM) and phosphate (Kd 26 microM). Ligand affinity increases by the factor 1.76 per an added (monomer unit from phosphate to d(pT) and then for oligonucleotides d(Tp)nT (n 1 to 19-20). At n greater than 19-20, the ligand affinity remained constant. The complete ethylation of phosphodiester groups lowers affinity of the oligothymidylates to the enzyme by approximately 10 times, and comparable decrease of Pt2+-oligonucleotide affinity to polymerase is caused by the absence of Mn2+-ions. The data obtained led to suggestion that one Me2+-dependent electrostatic contact of the template phosphodiester group with the enzyme takes place (delta G = -1.45...-1.75 kcal/mole). Formation of a hydrogen bond with the oxygen atom of P = O group of the same template phosphate is also assumed (delta G = -4.8...-4.9 kcal/mole). Other template internucleotide phosphates do not interact with the enzyme but the bases of oligonucleotides take part in hydrophobic interactions with the template binding site. Gibbs energy changes by -0.34 kcal/mole when the template is lengthened by one unit.     

Eukaryotic and prokaryotic DNA-polymerase. II. The role of internucleotide phosphate groups of a template in its binding with the enzyme

The affinity of different ligands (phosphate, nucleoside monophosphates, oligonucleotides) to the template binding site of DNA polymerase alpha from human placenta was estimated. To this goal, dependences of rate of the enzyme inactivation by the affinity reagent d(pT)2pC[Pt2+(NH3)2OH](pT)7 on the concentration of these ligands as competitive inhibitors were determined. Minimal ligands capable to bind with the template site of DNA polymerase alpha were shown to be triethylphosphate (Kd 600 microM) and phosphate (Kd 53 microM). Ligand affinity increases by the factor 1.71 per added monomer unit from phosphate to d(pT) and then for oligothymidylates d(Tp)nT (n 1 to 14). The partial ethylation of phosphodiester groups does not change the efficiency of the oligothymidylate binding with the enzyme. However, the complete ethylation of these groups lowers affinity of the oligothymidylates to the enzyme by 7-9 times. The decrease is comparable with the change of Pt2+-decathymidylate affinity to the enzyme caused by Mn2+-ions. The data obtained led to suggestion that an electrostatic contact (most likely, Me2+-dependent) of phosphodiester group with the enzyme takes place. The type of contact is confirmed by Gibbs' energy change 1.1-1.4 kcal/mole. Formation of a hydrogen bond with the oxygen atom of P = O group of the same phosphate is also assumed (delta G =--4.4 . . .--4.5 kcal/mole). The other internucleotide phosphates and all bases of oligonucleotides form neither hydrogen bonds nor electrostatic contacts with the template binding site. Gibbs' energy changes by 0.32 kcal/mole when the template is lengthened by one unit. We suppose that this value characterizes the energy gain in the transition of oligonucleotide template from aquous medium to the hydrophobic environement of the enzyme active site. Comparison of Km values of oligothymidylates and their partially or completely ethylated analogues as templates in the reaction of DNA polymerization catalysed by DNA polymerase alpha from human placenta and Klenow's fragment of E. coli DNA polymerase I suggests a similar mechanism of template recognition by both enzymes.     

RNA-dependent DNA-polymerase from avian myeloblastosis virus: effectiveness of interaction with oligothymidylate primers of various length]

Optimal conditions for the reaction of polymerization catalyzed by RNA-dependent DNA-polymerase from AMV on poly(A)- and poly(dA)-templates with d(pT)n-primers were established. Optimal concentrations of the components and pH of the reaction mixtures were found out to differ significantly. dTTP was shown to be both a nucleotide substrate and a minimal primer of the polymerization. The Km values for d(pT)2-primer (Km = 0.11 mM and 0.54 for poly(A) and poly(dA)-templates, respectively) and longer oligothymidylates were estimated. The lengthening of d(pT)n (n = 2-10) by one mononucleotide unit led to a 3-fold and 2-fold decrease of Km value for poly(A) and poly(dA), respectively. Further lengthening of the primer (n = 10-25) did not affect Km for the primers. The maximal rates of polymerization did not depend on primer length. The activation reaction (Ea = 12 kcal/mol) of polymerization on poly(A) was considerably lower than that on poly(dA) (Ea = 50 kcal/mol). In both cases a highly processive polymerization was observed. It was suggested that the synthesis had been more effective on poly(A)-template due to a more effective formation of the complex enzyme primer template.     

Distinction between the two basic mechanisms of cation transport in the cardiac Na(+)-Ca2+ exchange system

In order to distinguish between the Ping-Pong and sequential mechanisms of cation transport in the cardiac Na(+)-Ca2+ exchange system, the initial rates of the Nai-dependent 45Ca uptake (t = 1 s) were measured in reconstituted proteoliposomes, loaded with a Ca chelator. Under "zero-trans" conditions ([Na]o = [Ca]i = 0) at a fixed [Na]i = 10-160 mM with varying [45Ca]o = 2.5-122 microM for each [Na]i, the Km and Vmax values increased from 7.7 to 33.5 microM and from 2.3 to 9.0 nmol.mg-1.s-1, respectively. The Vmax/Km values show a +/- 2-10% deviation from the average value of 0.274 nmol.mg-1.s-1.microM-1 over the whole range of [Na]i. These deviations are within the standard error of Vmax (+/- 3-7%), Km (+/- 11-17%), and Vmax/Km (+/- 11-19%). This suggests that, under conditions in which Vmax and Km are [Na]i dependent and vary 4-5-fold, the Vmax/Km values are constant within the experimental error. In the presence of K(+)-valinomycin the Vmax/Km values are 0.85 +/- 0.17 and 1.08 +/- 0.18 nmol.mg-1.s-1.microM-1 at [Na]i = 20 and 160 mM, respectively, suggesting that under conditions of "short circuit" of the membrane potential the Vmax/Km values still exhibit the [Na]i independence. At a very low fixed [45Ca]o = 1.1 microM with varying [Na]i = 10-160 mM, the initial rates were found to be [Na]i independent. At a high fixed [45Ca]o = 92 microM the initial rates show a sigmoidal dependence on the [Na]i with Vmax = 13.8 nmol.mg-1.s-1, KmNa = 21 mM, and Hill coefficient nH = 1.5. The presented data support a Ping-Pong (consecutive) mechanism of cation transport in the Na(+)-Ca2+ exchanger.     

Site-specific 1,N6-ethenoadenylated single-stranded oligonucleotides as structural probes for the T4 gene 32 protein-ssDNA complex

Bacteriophage T4 gene 32 protein (g32P) is a DNA replication accessory protein that binds single-stranded (ss) nucleic acids nonspecifically, independent of nucleotide sequence. G32P contains 1 mol of Zn(II)/mol of protein monomer, which can be substituted with Co(II), with maintenance of the structure and activity of the molecule. The Co(II) is coordinated via approximately tetrahedral ligand symmetry by three Cys sulfur atoms and therefore exhibits intense S(-)----Co(II) ligand to metal charge-transfer (LMCT) transitions in the near ultraviolet [Giedroc, D. P., et al. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 8452-8456]. A series of fluorescent 1,N6-ethenoadenosine (epsilon A)-containing oligonucleotides conforming to the structure (5'----3') d[(Tp)m epsilon A(pT)l-m-1] where 0 less than or equal to m less than or equal to l - 1 and length (l) six or eight nucleotides have been evaluated as dynamics probes and potential fluorescence energy transfer donors to Co(II) in mapping the spatial proximity of the (fixed) intrinsic metal ion and a variably positioned epsilon A-base in a series of protein-nucleic acid complexes. We provide spectroscopic evidence that the epsilon A-oligonucleotides bind to g32P-(A + B) with a fixed polarity of the phosphodiester chain. A Trp side chain(s) makes close approach to a epsilon A base positioned toward the 3' end of a bound l = 8 oligonucleotide. Six oligonucleotides of l = 8 and m = 0, 1, 3, 5, 6, or 7 were investigated as energy transfer donors to Co(II) at 0.1 M NaCl, pH 8.1, 25 degrees C upon binding to Co(II)-substituted or Zn(II) g32P-(A + B), i.e., in the presence and absence of an energy acceptor, respectively. Detectable quenching of the epsilon A-fluorescence by the Co(II)-LMCT acceptors was found to occur in all epsilon A-oligonucleotide-protein complexes, yielding energy transfer efficiencies (E) of 0.43, 0.31, 0.26, 0.26, 0.28, and 0.41 for l = 8 and m = 0, 1, 3, 5, 6, and 7 epsilon A-oligonucleotides, respectively. The two-dimensional distances R (in A) were found to vary as follows: d[epsilon A(pT)7] (m = 0), 16.0 (15.5-16.9); d[Tp epsilon A(pT)6] (m = 1), 17.7 (16.9-19.1); d[(Tp)3 epsilon A(pT)4] (m = 3), 20.7 (19.5-22.1); d[(Tp)5 epsilon A(pT)2] (m = 5), 20.5 (19.5-21.9); d[(Tp)6 epsilon ApT] (m = 6), 19.0 (18.0-20.4); and d[(Tp)7 epsilon A] (m = 7), 18.6 (17.8-19.8).(ABSTRACT TRUNCATED AT 400 WORDS)     

Adenine base unstacking dominates the observed enthalpy and heat capacity changes for the Escherichia coli SSB tetramer binding to single-stranded oligoadenylates.

Isothermal titration calorimetry (ITC) was used to test the hypothesis that the relatively small enthalpy change (DeltaHobs) and large negative heat capacity change (DeltaCp,obs) observed for the binding of the Escherichia coli SSB protein to single-stranded (ss) oligodeoxyadenylates result from the temperature-dependent adenine base unstacking equilibrium that is thermodynamically coupled to binding. We have determined DeltaH1,obs for the binding of 1 mole of each of dT(pT)34, dC(pC)34, and dA(pA)34 to the SSB tetramer (20 mM NaCl at pH 8.1). For dT(pT)34 and dC(pC)34, we found large, negative values for DeltaH1,obs of -75 +/- 1 and -85 +/- 2 kcal/mol at 25 degrees C, with DeltaCp,obs values of -540 +/- 20 and -570 +/- 30 cal mol-1 K-1 (7-50 degrees C), respectively. However, for SSB-dA(pA)34 binding, DeltaH1,obs is considerably less negative (-14 +/- 1 kcal/mol at 25 degrees C), even becoming positive at temperatures below 13 degrees C, and DeltaCp,obs is nearly twice as large in magnitude (-1180 +/- 40 cal mol-1 K-1). These very different thermodynamic properties for SSB-dA(pA)34 binding appear to result from the fact that the bases in dA(pA)34 are more stacked at any temperature than are the bases in dC(pC)34 or dT(pT)34 and that the bases become unstacked within the SSB-ssDNA complexes. Therefore, the DeltaCp,obs for SSB-ssDNA binding has multiple contributions, a major one being the coupling to binding of a temperature-dependent conformational change in the ssDNA, although SSB binding to unstacked ssDNA still has an "intrinsic" negative DeltaCp,0. In general, such temperature-dependent changes in the conformational "end states" of interacting macromolecules can contribute significantly to both DeltaCp,obs and DeltaHobs.