Facile synthesis of a fluorescent deoxycytidine analogue suitable for probing the RecA nucleoprotein filament.

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]

Design and evaluation of a tryptophanless RecA protein with wild type activity.

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.

The stretched DNA geometry of recombination and repair nucleoprotein filaments.

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.

A perspective on biological catalysis.

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.

Molecular basis for nonadditive mutational effects in Escherichia coli dihydrofolate reductase.

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.

Nucleic acid hybridization: triplex stability and energetics.

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.

Equilibrium association constants for oligonucleotide-directed triple helix formation at single DNA sites: linkage to cation valence and concentration.

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)

Influence of pH on the equilibrium association constants for oligodeoxyribonucleotide-directed triple helix formation at single DNA sites.

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.

Thermodynamic characterization of the stability and the melting behavior of a DNA triplex: a spectroscopic and calorimetric study.

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.