Abstract: Signal transduction, regulatory processes and pharmaceutical responses are highly dependent upon ligand residence times. Gaining insight into how physical factors influence residence times (1/k(off)) should enhance our ability to manipulate biological interactions. We report experiments that yield structural insight into k(off) involving a series of eight 2,4-diaminopyrimidine inhibitors of dihydrofolate reductase whose binding affinities vary by six orders of magnitude. NMR relaxation-dispersion experiments revealed a common set of residues near the binding site that undergo a concerted millisecond-timescale switching event to a previously unidentified conformation. The rate of switching from ground to excited conformations correlates exponentially with the binding affinity K(i) and k(off), suggesting that protein dynamics serves as a mechanical initiator of ligand dissociation within this series and potentially for other macromolecule-ligand systems. Although the forward rate of conformational exchange, k(conf,forward), is faster than k(off), the use of the ligand series allowed for connections to be drawn between kinetic events on different timescales.
Abstract: Structure-based drug design relies on static protein structures despite significant evidence for the need to include protein dynamics as a serious consideration. In practice, dynamic motions are neglected because they are not understood well enough to model, a situation resulting from a lack of explicit experimental examples of dynamic receptor-ligand complexes. Here, we report high-resolution details of pronounced ~1 ms time scale motions of a receptor-small molecule complex using a combination of NMR and X-ray crystallography. Large conformational dynamics in Escherichia coli dihydrofolate reductase are driven by internal switching motions of the drug-like, nanomolar-affinity inhibitor. Carr-Purcell-Meiboom-Gill relaxation dispersion experiments and NOEs revealed the crystal structure to contain critical elements of the high energy protein-ligand conformation. The availability of accurate, structurally resolved dynamics in a protein-ligand complex should serve as a valuable benchmark for modeling dynamics in other receptor-ligand complexes and prediction of binding affinities.
Abstract: Abstract The activities of the bacterial RecA protein are involved in the de novo development and transmission of antibiotic resistance genes, thus allowing bacteria to overcome the metabolic stress induced by antibacterial agents. RecA is ubiquitous and highly conserved among bacteria, but has only distant homologs in human cells. Together, this evidence points to RecA as a novel and attractive antibacterial drug target. All known RecA functions require the formation of a complex formed by multiple adenosine 5'-O-triphosphate (ATP)-bound RecA monomers on single-stranded DNA. In this complex, RecA hydrolyzes ATP. Although several methods for assessing RecA's ATPase activity have been reported, these assay conditions included relatively high concentrations of enzyme and ATP and thereby restricted the RecA conformational state. Herein, we describe the validation of commercial reagents (Transcreener(®) adenosine 5'-O-diphosphate [ADP](2) fluorescence polarization assay) for the high-throughput measurement of RecA's ATPase activity with lower concentrations of ATP and RecA. Under optimized conditions, ADP detection by the Transcreener reagent provided robust and reproducible activity data (Z'=0.92). Using the Transcreener assay, we screened 113,477 small molecules against purified RecA protein. In total, 177 small molecules were identified as confirmed hits, of which 79 were characterized by IC(50) values ≤10 μM and 35 were active in bioassays with live bacteria. This set of compounds comprises previously unidentified scaffolds for RecA inhibition and represents tractable hit structures for efforts aimed at tuning RecA inhibitory activity in both biochemical and bacteriological assays.
Abstract: The bacterial RecA protein has been implicated as a bacterial drug target not as an antimicrobial target, but as an adjuvant target with the potential to suppress the mechanism by which bacteria gain drug resistance. In order to identify small molecules that inhibit RecA/ssDNA nucleoprotein filament formation, we have adapted the phosphomolybdate-blue ATPase assay for high throughput screening to determine RecA ATPase activity against a library of 33,600 compounds, which is a selected representation of diverse structure of 350,000. Four distinct chemotypes were represented among the 40 validated hits. SAR and further chemical synthesis is underway to optimize this set of inhibitors to be used as antimicrobial adjuvant agents.
Abstract: The phenomenon of antibiotic resistance has created a need for the development of novel antibiotic classes with nonclassical cellular targets. Unfortunately, target-based drug discovery against proteins considered essential for in vitro bacterial viability has yielded few new therapeutic classes of antibiotics. Targeting the large proportion of genes considered nonessential that have yet to be explored by high-throughput screening, for example, RecA, can complement these efforts. Recent evidence suggests that RecA-controlled processes are responsible for tolerance to antibiotic chemotherapy and are involved in pathways that ultimately lead to full-fledged antibiotic resistance. Therefore inhibitors of RecA may serve as therapeutic adjuvants in combination chemotherapy of bacterial infectious diseases. Toward the goal of validating RecA as a novel target in the chemotherapy of bacterial infections, the authors have screened 35,780 small molecules against RecA. In total, 80 small molecules were identified as primary hits and could be clustered in 6 distinct chemotype clades. The most potent class of hits was further examined, and 1 member compound was found to inhibit RecA-mediated strand exchange and prevent ciprofloxacin-induced SOS expression in Escherichia coli. This compound represents the first small molecule demonstrating an ability to inhibit the bacterial SOS response in live bacterial cell cultures.
Abstract: Bacterial RecA promotes the development and transmission of antibiotic resistance genes by self-assembling into an ATP-hydrolyzing filamentous homopolymer on single-stranded DNA. We report the design of a 29mer peptide based on the RecA N-terminal domain involved in intermonomer contact that inhibits RecA filament assembly with an IC50 of 3 microM.
Abstract: The roles of bacterial RecA in the evolution and transmission of antibiotic resistance genes make it an attractive target for inhibition by small molecules. We report two complementary fluorescence-based ATPase assays that were used to screen for inhibitors of RecA. We elected to employ the ADP-linked variation of the assay, with a Z' factor of 0.83 in 96-well microplates, to assess whether 18 select compounds could inhibit ATP hydrolysis by RecA. The compounds represented five sets of related inhibitor scaffolds, each of which had the potential to cross-inhibit RecA. Although nucleotide analogs, known inhibitors of GHL ATPases, and known protein kinase inhibitors were not active against RecA, we found that three suramin-like agents substantially inhibited RecA's ATPase activity.
Abstract: The bacterial RecA protein has been implicated in the evolution of antibiotic resistance in pathogens, which is an escalating problem worldwide. The discovery of small molecules that can selectively modulate RecA's activities can be exploited to tease apart its roles in the de novo development and transmission of antibiotic resistance genes. Toward the goal of discovering small-molecule ligands that can prevent either the assembly of an active RecA-DNA filament or its subsequent ATP-dependent motor activities, we report the design and initial validation of a pair of rapid and robust screening assays suitable for the identification of inhibitors of RecA activities. One assay is based on established methods for monitoring ATPase enzyme activity and the second is a novel assay for RecA-DNA filament assembly using fluorescence polarization. Taken together, the assay results reveal complementary sets of agents that can either suppress selectively only the ATP-driven motor activities of the RecA-DNA filament or prevent assembly of active RecA-DNA filaments altogether. The screening assays can be readily configured for use in future automated high-throughput screening projects to discover potent inhibitors that may be developed into novel adjuvants for antibiotic chemotherapy that moderate the development and transmission of antibiotic resistance genes and increase the antibiotic therapeutic index.
Abstract: The RecA protein of Escherichia coli plays a crucial roles in DNA recombination and repair, as well as various aspects of bacterial pathogenicity. The formation of a RecA-ATP-ssDNA complex initiates all RecA activities and yet a complete structural and mechanistic description of this filament has remained elusive. An analysis of RecA-DNA interactions was performed using fluorescently labeled oligonucleotides. A direct comparison was made between fluorescein and several fluorescent nucleosides. The fluorescent guanine analog 6-methylisoxanthopterin (6MI) demonstrated significant advantages over the other fluorophores and represents an important new tool for characterizing RecA-DNA interactions.
Abstract: The Escherichia coli RecA protein is the prototype of a class of proteins playing a central role in genomic repair and recombination in all organisms. The unresolved mechanistic strategy by which RecA aligns a single strand of DNA with a duplex DNA and mediates a DNA strand switch is central to understanding its recombinational activities. Toward a molecular-level understanding of RecA-mediated DNA strand exchange, we explored its mechanism using oligonucleotide substrates and the intrinsic fluorescence of 6-methylisoxanthopterin (6MI). Steady- and presteady-state spectrofluorometric data demonstrate that the reaction proceeds via a sequential four-step mechanism comprising a rapid, bimolecular association step followed by three slower unimolecular steps. Previous authors have proposed multistep mechanisms involving two or three steps. Careful analysis of the differences among the experimental systems revealed a previously undiscovered intermediate (N1) whose formation may be crucial in the kinetic discrimination of homologous and heterologous sequences. This observation has important implications for probing the fastest events in DNA strand exchange using 6MI to further elucidate the molecular mechanisms of recombination and recombinational repair.
Abstract: The roles of the RecA protein in the survival of bacteria and the evolution of resistance to antibiotics make it an attractive target for inhibition by small molecules. The activity of RecA is dependent on the formation of a nucleoprotein filament on single-stranded DNA that hydrolyzes ATP. We probed the nucleotide binding site of the active RecA protein using modified nucleotide triphosphates to discern key structural elements of the nucleotide and of the binding site that result in the activation of RecA for NTP hydrolysis. Our results show that the RecA-catalyzed hydrolysis of a given nucleotide triphosphate or analogue thereof is exquisitely sensitive to certain structural elements of both the base and ribose moieties. Furthermore, our ligand-based approach to probing the RecA ATP binding site indicated that the binding of nucleotides by RecA was found to be conformationally selective. Using a binding screen that can be readily adapted to high-throughput techniques, we were able to segregate nucleotides that interact with RecA into two classes: (1) NTPs that preferentially bind the active nucleoprotein filament conformation and either serve as substrates for or competitively inhibit hydrolysis and (2) nonsubstrate NTPs that preferentially bind the inactive RecA conformation and facilitate dissociation of the RecA-DNA species. These results are discussed in the context of a recent structural model for the active RecA nucleoprotein filament and provide us with important information for the design of potent, conformationally selective modulators of RecA activities.
Abstract: Despite intense effort over the past 30 years, the molecular determinants of sequence selectivity in RecA-mediated homologous recombination have remained elusive. Here, we describe when and how sequence homology is recognized between DNA strands during recombination in the context of a kinetic model for RecA-mediated DNA strand exchange. We characterized the transient intermediates of the reaction using pre-steady-state kinetic analysis of strand exchange using oligonucleotide substrates containing a single fluorescent G analog. We observed that the reaction system was sensitive to heterology between the DNA substrates; however, such a "heterology effect" was not manifest when functional groups were added to or removed from the edges of the base-pairs facing the minor groove of the substrate duplex. Hence, RecA-mediated recombination must occur without the involvement of a triple helix, even as a transient intermediate in the process. The fastest detectable reaction phase was accelerated when the structure or stability of the substrate duplex was perturbed by internal mismatches or the replacement of G.C by I.C base-pairs. These findings indicate that the sequence specificity in recombination is achieved by Watson-Crick pairing in the context of base-pair dynamics inherent to the extended DNA structure bound by RecA during strand exchange.
Abstract: The Escherichia coli RecA protein is the prototype of a class of proteins that play central roles in genomic repair and recombination in all organisms. The unresolved mechanistic strategy by which RecA aligns a single strand of DNA with a duplex DNA and mediates a DNA strand switch is central to understanding homologous recombination. We explored the mechanism of RecA-mediated DNA-strand exchange using oligonucleotide substrates with the intrinsic fluorophore 6-methylisoxanthopterin. Pre-steady-state spectrofluorometric analysis elucidated the earliest transient intermediates formed during recombination and delineated the mechanistic strategy by which RecA facilitates this process. The structural features of the first detectable intermediate and the energetic characteristics of its formation were consistent with interactions between a few bases of the single-stranded DNA and the minor groove of a locally melted or stretched duplex DNA. Further analysis revealed RecA to be an unusual enzyme in that entropic rather than enthalpic contributions dominate its catalytic function, and no unambiguously active role for the protein was detected in the earliest molecular events of recombination. The data best support the conclusion that the mechanistic strategy of RecA likely relies on intrinsic DNA dynamics.
Abstract: The Escherichia coli RecA protein is the prototypical member of a family of molecular motors that transduces ATP binding and hydrolysis for mechanical function. While many general mechanistic features of RecA action are known, specific structural and functional insights into the molecular basis of RecA activation remain elusive. Toward a more complete understanding of the interdependence between ATP and DNA binding by RecA, we report the characterization of a mutant RecA protein wherein the aspartate residue at position 100 within the ATP binding site is replaced by arginine. Physiologically, D100R RecA was characterized by an inducible, albeit reduced, activation of the SOS response and a diminished ability to promote cellular survival after UV irradiation. Biochemically, the D100R substitution caused a surprisingly modest perturbation of RecA-ATP interactions and an unexpected and significant decrease in the affinity of RecA for ssDNA. Moreover, in vitro assays of RecA activities requiring the coordinated processing of ATP and DNA revealed (1) a 2-5-fold decrease in steady-state turnover of ATP; (2) no formation of mixed nucleoprotein filaments when wild-type and D100R RecA compete for limiting ssDNA; and (3) no formation of strand exchange reaction products. Taken together, these results suggest that the D100R mutational effects on isolated RecA activities combine synergistically to perturb its higher-order functions. We conclude that the replacement of Asp100 resulted in a change in the electrostatic complementarity between RecA monomers during active filament assembly that prevents the protein from fully accessing the active multimeric state.
Abstract: We report that N(6)-(1-naphthyl)-ADP inhibits the Escherichia coli RecA protein in vitro. A novel rapid screen identified it as a potent inhibitor of RecA nucleoprotein filament formation, and further characterization established it as an ATP-competitive inhibitor of RecA-catalyzed ATP hydrolysis. This and other inhibitors of RecA activities represent a new approach for understanding the molecular targets and pathways involved in the evolution of antibiotic resistance in bacteria.
Abstract: In bacteria, the RecA protein plays important roles in a number of DNA recombination and repair processes, including homologous recombination, SOS induction and recombinational DNA repair. We have explored the idea that the Escherichia coli RecA protein's functions could be controlled by small molecules. We investigated the 2:1 complex of zinc(II) with 1,4-dithio-l-threitol (l-DTT) that inhibits the E. coli rho transcription terminator, which is a hexameric ATP motor protein and is structurally homologous to RecA. We found that both the complex and ZnCl(2) inhibit the single-stranded DNA-dependent ATPase activity of RecA at sub-millimolar concentrations. Investigation of a variety of metal dications (0.4 mM final concentration) determined that zinc(II), copper(II) and mercury(II) all induce the precipitation of RecA, while the dichloride salts of calcium, manganese, barium, cobalt, and nickel do not. The inhibition of RecA activity by Zn(II), Cu(II) and Hg(II) results from the metal-dependent initiation of RecA aggregation. These observations may have implications for the design of biophysical experiments requiring solid-phase RecA protein, for a more complete understanding of metal toxicities, and for the design of metal-chelate inhibitors of prokaryotic DNA repair.
Abstract: The RecA protein of Escherichia coli controls the SOS response for DNA damage tolerance and plays a crucial role in recombinational DNA repair. The formation of a RecA.ATP.ssDNA complex initiates all RecA activities, and yet this process is not understood at the molecular level. An analysis of RecA.DNA interactions was performed using both a mutant RecA protein containing a tryptophan (Trp) reporter and oligodeoxyribonucleotides (ODNs) containing a fluorescent guanine analogue, 6-methylisoxanthopterin (6MI). Experiments using fluorescent ODNs allowed structurally distinct nucleoprotein filaments, formed in the absence and presence of ATPgammaS (a slowly hydrolyzed analogue of ATP), to be differentiated directly. Stopped-flow spectrofluorometry, combined with presteady-state kinetic analyses, revealed unexpected differences in the rates of RecA.ODN and RecA.ATPgammaS.ODN complex assembly. This is the first demonstration that such intrinsically fluorescent synthetic DNAs can be used to characterize definitively the real-time assembly and activation of RecA.ssDNA complexes. Surprisingly, the ssDNA binding event is almost 50-fold slower in the presence of the activating ATPgammaS cofactor. Furthermore, a combination of time-dependent emission changes from 6MI and Trp allowed the first direct chemical test of whether an inactive filament can isomerize to the active state. The results revealed that, unlike the hexameric motor proteins, the inactive RecA filament cannot directly convert to the active state upon ATPgammaS binding. These results have implications for understanding how a coincidence of functions--an ATP-communicated signal-like activity and an ATP-driven motorlike activity--are resolved within a single protein molecule.
Abstract: [structure: see text] We describe the synthesis of a chain-terminating mRNA cap dinucleotide and its use in the in vitro transcription of homogeneously capped RNA. Computer modeling strongly indicates that RNA capped with the new compound will be a substrate for cap-dependent translation.
Abstract: The RecA protein of Escherichia coli plays important roles in homologous recombination, recombinational DNA repair, and SOS induction. Because its functions are conserved among the phylogenetic kingdoms, RecA investigations have provided a paradigm for understanding these biological processes. The RecA protein has been overproduced in E. coli and purified using a variety of purification schemes requiring multiple, time-intensive steps. The purification schemes share a dependence on appropriate RecA structure and/or function at one or more steps. In this report, we used a modified protein splicing element (intein) and a chitin-binding domain, fused to the C-terminus of RecA, to facilitate a one-step affinity purification of RecA protein without modification of the native protein sequence. Following the single chromatographic step, RecA protein that is greater than 95% physical purity at a concentration of greater than microM was obtained. The protein displays in vitro activities that are identical to those of protein isolated using classical procedures. The purification strategy described here promises to yield mutant RecA proteins in sufficient quantity for rigorous biophysical characterization without dependence on intrinsic RecA function.
Abstract: The RecA protein of Escherichia coli plays essential roles in homologous recombination and restarting stalled DNA replication forks. In vitro, the protein mediates DNA strand exchange between single-stranded (ssDNA) and homologous double-stranded DNA (dsDNA) molecules that serves as a model system for the in vivo processes. To date, no high-resolution structure of the key intermediate, comprised of three DNA strands simultaneously bound to a RecA filament (RecA-tsDNA complex), has been reported. We present a systematic characterization of the helical geometries of the three DNA strands of the RecA-tsDNA complex using fluorescence resonance energy transfer (FRET) under physiologically relevant solution conditions. FRET donor and acceptor dyes were used to label different DNA strands, and the interfluorophore distances were inferred from energy transfer efficiencies measured as a function of the base-pair separation between the two dyes. The energy transfer efficiencies were first measured on a control RecA-dsDNA complex, and the calculated helical parameters (h approximately 5 A, Omega(h) approximately 20 degrees ) were consistent with structural conclusions derived from electron microscopy (EM) and other classic biochemical methods. Measurements of the helical parameters for the RecA-tsDNA complex revealed that all three DNA strands adopt extended and unwound conformations similar to those of RecA-bound dsDNA. The structural data are consistent with the hypothesis that this complex is a late, post-strand-exchange intermediate with the outgoing strand shifted by about three base-pairs with respect to its registry with the incoming and complementary strands. Furthermore, the bases of the incoming and complementary strands are displaced away from the helix axis toward the minor groove of the heteroduplex, and the bases of the outgoing strand lie in the major groove of the heteroduplex. We present a model for the strand exchange intermediate in which homologous contacts preceding strand exchange arise in the minor groove of the substrate dsDNA.
Abstract: The C-terminal domain of the Escherichia coli RecA protein contains two tryptophan residues whose native fluorescence emission provides an interfering background signal when other fluorophores such as 1,N(6)-ethenoadenine, 2-aminopurine and other tryptophan residues are used to probe the protein's activities. Replacement of the wild type tryptophans with nonfluorescent residues is not trivial because one tryptophan is highly conserved and the C-terminal domain functions in both DNA binding as well as interfilament protein-protein contact. We undertook the task of creating a tryptophanless RecA protein with WT RecA activity by selecting suitable amino acid replacements for Trp290 and Trp308. Mutant proteins were screened in vivo using assays of SOS induction and cell survival following UV irradiation. Based on its activity in these assays, the W290H-W308F W-less RecA was purified for in vitro characterization and functioned like WT RecA in DNA-dependent ATPase and DNA strand exchange assays. Spectrofluorometry indicates that the W290H-W308F RecA protein generates no significant emission when excited with 295-nm light. Based on its ability to function as wild type protein in vivo and in vitro, this dark RecA protein will be useful for future fluorescence experiments.
Abstract: We report the synthesis of the fluorescent 2'-deoxycytidine analogue 5-methylpyrimidin-2-one nucleoside, its incorporation at three specified sites in a single 60-nucleotide DNA molecule, and the use of its total and polarized intrinsic fluorescence to characterize RecA-DNA complexes. [reaction: see text]
Abstract: The RecA protein of Escherichia coli plays essential roles in homologous recombination and restarting stalled DNA replication forks. In vitro, the protein mediates DNA strand exchange between single-stranded (ssDNA) and homologous double-stranded DNA (dsDNA) molecules that serves as a model system for the in vivo processes. To date, no high-resolution structure of the key intermediate, comprised of three DNA strands simultaneously bound to a RecA filament (RecA x tsDNA complex), has been elucidated by classical methods. Here we review the systematic characterization of the helical geometries of the three DNA strands of the RecA x tsDNA complex using fluorescence resonance energy transfer (FRET) under physiologically relevant solution conditions. Measurements of the helical parameters for the RecA x tsDNA complex are consistent with the hypothesis that this complex is a late, poststrand-exchange intermediate with the outgoing strand shifted by about three base pairs with respect to its registry with the incoming and complementary strands. All three strands in the RecA x tsDNA complex adopt extended and unwound conformations similar to those of RecA-bound ssDNA and dsDNA.
Abstract: We have analysed enzyme catalysis through a re-examination of the reaction coordinate. The ground state of the enzyme-substrate complex is shown to be related to the transition state through the mean force acting along the reaction path; as such, catalytic strategies cannot be resolved into ground state destabilization versus transition state stabilization. We compare the role of active-site residues in the chemical step with the analogous role played by solvent molecules in the environment of the noncatalysed reaction. We conclude that enzyme catalysis is significantly enhanced by the ability of the enzyme to preorganize the reaction environment. This complementation of the enzyme to the substrate's transition state geometry acts to eliminate the slow components of solvent reorganization required for reactions in aqueous solution. Dramatically strong binding of the transition state geometry is not required.
Abstract: In this chapter, we review the current state of the thermodynamic database for triple helical oligonucleotide hybridization reactions and present a critical assessment of the methods used to obtain the relevant data. The thermodynamic stability of triple-helix oligonucleotide constructs is discussed in terms of its dependence on temperature, chain length, pH, salt, base sequence, base and backbone modifications, and ligand binding. In particular, we examine the coupling of hybridization equilibria to proton, cation, and drug-binding equilibria. Throughout the chapter, we emphasize that a detailed understanding of the endogenous and exogenous variables that control triplex stability is required for the rational design of oligonucleotides for specific therapeutic, diagnostic, and/or biotechnological applications, as well as for elucidating the potential cellular roles of these higher-order nucleic acid complexes.
Abstract: Recently, two sets of single, double, and quadruple residue changes within the hydrophobic substrate binding pocket of Escherichia coli dihydrofolate reductase (5,6,7,8-tetrahydrofolate+ oxidoreductase, EC 1.5.1.3) were shown to exhibit nonadditive mutational effects [Huang, Z., Wagner, C. R., & Benkovic, S. J. (1994) Biochemistry 33, 11576--11585]. In particular, the analysis of data for the L28Y, L54F, and L28Y-L54F mutations revealed nonadditive changes in the free energy associated with the substrate and cofactor binding, hydride transfer, and product release steps. Construction of a related set of mutant proteins including L28F and L28F-L54F permits a comparison of similar energy changes and provides a means for assessing differences in the interactions of Phe28 and Tyr28 with both the ligands and the side chains at residue 54. We find a single functional group change, from Phe C4-H to Tyr C4-OH, can influence the additivity of mutational effects and serve as a probe to monitor the appearance of differing enzyme conformations along the reaction pathway through changes in the interaction energy (delta GI). The comparison of additivity/nonadditivity in free energy changes for three interrelated double mutational cycles (WT --> L28F-L54F, WT --> L28Y-L54F, and L28F --> l28Y-L54F) demonstrates that the side chains of positions 28 and 54 interact cooperatively to facilitate hydride transfer by preferentially influencing the enzyme--substrate ground-state complexes. The delta GI data for individual steps also provide evidence for multiple conformations of the enzyme operating during the catalytic cycle. The fact that there are no published examples of the synergistic enhancement of favorable mutational effects is consistent with the expectation that the binding/active site surface of wild-type dihydrofolate reductase has been optimized.
Abstract: The linkage between the energetics of oligonucleotide-directed triple helix formation and the cationic solution environment has been investigated in mixed-valence salt solutions. Equilibrium constants for formation of the local pyrimidine.purine.pyrimidine structure afforded by binding of the oligonucleotide 5'-d(T*TTTTCTCTCTCTCT)-3' to a single site within a 339-bp plasmid fragment were measured using quantitative affinity cleavage titrations at pH 7.0 and 22 degrees C in the presence of various concentrations of KCl, MgCl2, and spermine tetrahydrochloride (SpmCl4). In a solution containing 10 mM NaCl, 140 mM KCl, 1.0 mM MgCl2, and 1.0 mM SpmCl4, the measured binding constant was 3.3 (+/- 1.4) x 10(5) M-1. The equilibrium constant previously reported for the same association reaction in 100 mM NaCl and 1 mM SpmCl4 at the same temperature and pH was 10-fold higher [Singleton, S. F., & Dervan, P. B. (1992) J. Am. Chem. Soc. 114, 6957-6965]. Further study demonstrated that varying the potassium ion concentration between 5.0 and 140 mM (in the presence of 10 mM NaCl, 1.0 mM MgCl2, and 1.0 mM SpmCl4) resulted in an overall 100-fold decrease in the binding affinity from the lowest to the highest concentration. In contrast, measured binding constants increased 500-fold as the spermine concentration was increased from 0.40 to 4.0 mM (in the presence of 10 mM NaCl, 140 mM KCl, and 1.0 mM MgCl2). There was a modest effect on the binding constant (a 3-fold decrease) upon varying the magnesium ion concentration from 0.10 to 10 mM (in the presence of 10 mM NaCl, 140 mM KCl, and 1.0 mM SpmCl4).(ABSTRACT TRUNCATED AT 250 WORDS)
Abstract: The energetics of oligodeoxyribonucleotide-directed triple helix formation for the pyrimidine.purine.pyrimidine structural motif were determined over the pH range 5.8-7.6 at 22 degrees C (100 mM Na+ and 1 mM spermine) using quantitative affinity cleavage titration. The equilibrium binding constants for 5'-TTTTTCTCTCTCTCT-3' (1) and 5'-TTTTTm5CTm5CTm5CTm5CTm5CT-3' (2, m5C is 2'-deoxy-5-methylcytidine) increased by 10- and 20-fold, respectively, from pH 7.6 to 5.8, indicating that the corresponding triple-helical complexes are stabilized by 1.4 and 1.7 kcal.mol-1, respectively, at the lower pH. Replacement of the five cytosine residues in 1 with 5-methylcytosine residues to yield 2 affords a stabilization of the triple helix by 0.1-0.4 kcal.mol-1 over the pH range 5.8-7.6. An analysis of these data in terms of a quantitative model for a general pH-dependent equilibrium transition revealed that pyrimidine oligonucleotides with cytidine and 5-methylcytidine form local triple-helical structures with apparent pKa's of 5.5 (C+GC triplets) and 5.7 (m5C+GC triplets), respectively, and that the oligonucleotides should bind to single sites on large DNA with apparent affinity constants of approximately 10(6) M-1 even above neutral pH.
Abstract: We report a complete thermodynamic characterization of the stability and the melting behavior of an oligomeric DNA triplex. The triplex chosen for study forms by way of major-groove Hoogsteen association of an all-pyrimidine 15-mer single strand (termed y15) with a Watson-Crick 21-mer duplex composed of one purine-rich strand (termed u21) and one pyrimidine-rich strand (termed y21). We find that the near-UV CD spectrum of the triplex can be duplicated by the addition of the B-like CD spectrum of the isolated 21-mer duplex and the CD spectrum of the 15-mer single strand. Spectroscopic and calorimetric measurements show that the triplex (y15.u21.y21) melts by two well-resolved sequential transitions. The first transition (melting temperature, Tm, approximately 30 degrees C) is pH-dependent and involves the thermal expulsion of the 15-mer strand to form the free duplex u21.y21 and the free single strand y15. The second transition (Tm approximately 65 degrees C) is pH-independent between pH 6 and 7 and reflects the thermal disruption of the u21.y21 Watson-Crick duplex to form the component single strands. The thermal stability of the y15.u21.y21 triplex increases with increasing Na+ concentration but is nearly independent of DNA strand concentration. Differential scanning calorimetric measurements at pH 6.5 show the triplex to be enthalpically stabilized by only 2.0 +/- 0.1 kcal/mol of base triplets (1 cal = 4.184 J), whereas the duplex is stabilized by 6.3 +/- 0.3 kcal/mol of base pairs. From the calorimetric data, we calculate that at 25 degrees C the y15.u21.y21 triplex is stabilized by a free energy of only 1.3 +/- 0.1 kcal/mol relative to its component u21.y21 duplex and y15 single strand, whereas the 21-mer duplex is stabilized by a free energy of 17.2 +/- 1.2 kcal/mol relative to its component single strands. The y15 single strand modified by methylation of cytosine at the C-5 position forms a triplex with the u21.y21 duplex, which exhibits enhanced thermal stability. The spectroscopic and calorimetric data reported here provide a quantitative measure of the influence of salt, temperature, pH, strand concentration, and base modification on the stability and the melting behavior of a DNA triplex. Such information should prove useful in designing third-strand oligonucleotides and in defining solution conditions for the effective use of triplex structure formation as a tool for modulating biochemical events.
