Base-paired Duplex Research Articles

Solution Structure of a Nonpolar, Non-Hydrogen-Bonded Base Pair Surrogate in DNA.

We describe the structure in aqueous solution of a DNA duplex containing a base pair that is structurally analogous to A-T but which lacks hydrogen bonds. Base analogues F (a nonpolar isostere of thymine) and Z (a nonpolar isostere of adenine) are paired opposite one another in a 12 base pair duplex. The sequence context is the binding site of recently studied transcription factor hSRY. The Z-F pair has been shown to be replicated surprisingly well and selectively by DNA polymerase enzymes, considering that it is destabilizing and lacks Watson-Crick hydrogen bonds. The enzymatic studies led to the suggestion that part of the functional activity arises because the pair resembles a natural one in geometry. The present results show that, despite the absence of Watson-Crick hydrogen bonds, the Z-F pair structurally resembles an A-T pair in the same context. This lends support to the proposal that shape matching is an important component in replication, and suggests the general utility of using Z-F as a nonpolar replacement for A-T in probing protein-DNA interactions.

A TOPRIM domain in the crystal structure of the catalytic core of Escherichia coli primase confirms a structural link to DNA topoisomerases

Primases synthesize short RNA strands on single-stranded DNA templates, thereby generating the hybrid duplexes required for the initiation of synthesis by DNA polymerases. We present the crystal structure of the catalytic unit of a primase enzyme, that of a ∼320 residue fragment of Escherichia coli primase, determined at 2.9 Å resolution. Central to the catalytic unit is a TOPRIM domain that is strikingly similar in its structure to that of corresponding domains in DNA topoisomerases, but is unrelated to the catalytic centers of other DNA or RNA polymerases. The catalytic domain of primase is crescent-shaped, and the concave face of the crescent is predicted to accommodate about 10 base-pairs of RNA-DNA duplex in a loose interaction, thereby limiting processivity.

Thermodynamic, spectroscopic, and equilibrium binding studies of DNA sequence context effects in four 40 base pair deoxyoligonucleotides.

Effects of different end sequences on melting, circular dichroism spectra (CD), and enzyme binding properties were investigated for four 40 base pair, non-self-complementary duplex DNA oligomers. The center sequences of these oligoduplexes have either of two 22 base pair modules flanked on both sides by sequences differing in AT content. Temperature-induced melting transitions monitored by differential scanning calorimetry (DSC) and ultraviolet absorbance were measured for the six duplexes in buffered 115 mM Na(+) solutions. Values of the melting transition enthalpy, DeltaH(cal), and entropy, DeltaS(cal), were obtained directly from DSC experiments. Melting transition parameters, DeltaH(vH) and DeltaS(vH), were also estimated from a van't Hoff analysis of optical melting curves collected as a function of DNA concentration, assuming that the melting transition is two-state. Melting free energies (20 degrees C) evaluated from DSC melting experiments on the four duplex DNAs ranged from -52.2 to -77.5 kcal/mol. Free energies based on the van't Hoff analysis were -37.9 to -58.8 kcal/mol. Although the values are different, trends in the melting free energies of the four duplex DNAs as a function of sequence were identical in both DSC and optical analyses. Subject to several assumptions, values for the initiation free energy were estimated for each duplex, defined as DeltaG(int) = DeltaG(cal) - DeltaG(pred), where DeltaG(cal) is the experimental free energy at 20 degrees C determined from the experimentially measured values of the transition enthalpy, DeltaH(cal), and entropy, DeltaS(cal). The predicted free energy of the sequence, DeltaG(pred)(20 degrees C), is obtained using published nearest-neighbor sequence stability values. For three of the four duplexes, values of DeltaG(int) are essentially nil. In contrast, the duplex with 81.8% GC has a considerably higher estimate of DeltaG(int) = 7.1 kcal/mol. The CD spectra for the six duplexes collected over the wavelength range from 200 to 320 nm are also sequence-dependent. Factor analysis of the CD spectra by singular value decomposition revealed that the experimental CD spectra could be reconstructed from linear combinations of two minor and one major subspectra. Changes in the coefficients of the major subspectrum for different sequences reflect incremental sequence-dependent variations of the CD spectra. Equilibrium binding by BamHI restriction endonuclease to the 40 base pair DNAs whose central eight base pairs contain the recognition sequence for BamHI restriction enzyme bounded by A.T base pairs, 5'-A-GGATCC-A-3' was investigated. Binding assays were performed by titering BamHI against a constant concentration of each of the duplex DNA substrates, in the absence of Mg(2+), followed by analysis by gel retardation. Under the conditions employed, the enzyme binds but does not cleave the DNAs. Results of the assays revealed two binding modes with retarded gel mobilities. Binding isotherms for the fraction of bound DNA species versus enzyme concentration for each binding mode were constructed and analyzed with a simple two-step equilibrium binding model. This analysis provided semiquantitative estimates on the equilibrium binding constants for BamHI to the four DNAs. Values obtained for the binding constants varied only 7-fold and ranged from 6 x 10(-)(8) to 42 x 10(-)(8) M, with binding free energies from -8.6 to -9.7 (+/- 0.2) kcal/mol depending on the sequence that flanks the enzyme binding site. Unlike what was found earlier in binding studies of the 22 base pair duplexes that constitute the core modules of the present 40-mers [Riccelli, P. V., Vallone, P. M., Kashin, I., Faldasz, B. D., Lane, M. J., and Benight, A. S. (1999) Biochemistry 38, 11197-11208], no obvious relationship between binding and stability was found for these longer DNAs. Apparently, effects of flanking sequence stability on restriction enzyme binding may only be measurable in very short duplex deoxyoligonucl

The crystal structure of the octamer [r(guauaca)dC]2 with six Watson-Crick base-pairs and two 3′ overhang residues

The crystal structure of an alternating RNA octamer, r(guauaca)dC (RNA bases are in lower case while the only DNA base is in upper case), with two 3′ overhang residues one of them a terminal deoxycytosine and the other a ribose adenine, has been determined at 2.2 Å resolution. The refined structure has an Rwork 18.6 % and Rfree 26.8 %. There are two independent duplexes (molecules I and II) in the asymmetric unit cell, a = 24.95, b = 45.25 and c = 73.67 Å, with space group P212121. Instead of forming a blunt end duplex with two a+·c mispairs and six Watson-Crick base-pairs, the strands in the duplex slide towards the 3′ direction forming a two-base overhang (radC) and a six Watson-Crick base-paired duplex. The duplexes are bent (molecule I, 20°; molecule II, 25°) and stack head-to-head to form a right-handed superhelix. The overhang residues are looped out and the penultimate adenines of the two residues at the top end (A15) are anti and at the bottom (A7) end are syn. The syn adenine bases form minor groove A∗(G·C) base triples with C8-H···N2 hydrogen bonds. The anti adenine in molecule II also forms a triple and a different C2-H···N3 hydrogen bond, while the other anti adenine in molecule I does not, it stacks on the looped out overhang base dC. The 3′ terminal deoxycytosines form two stacked hemiprotonated trans d(C·C)+ base-pairs and the pseudo dyad related molecules form four consecutive deoxyribose and ribose zipper hydrogen bonds in the minor groove.

Crystal Structure of an RNA Duplex [r(gugcaca)dC]2 with 3′-Dinucleoside Overhangs Forming a Superhelix

Crystal structure of the RNA octamer duplex, [r(gugcaca)dC] 2, with space group I212121 and the cell constants a=24.29, b=45.25 and c=73.68Å, has been determined and refined. The structural and packing architecture of the molecule consist of a highly bent six base paired duplex forming a right-handed superhelix stacked in tandem compared to an infinite pseudo- continuous column as is usually present in RNA crystal structures. The super helix could be formed by the head-to-head stacking (g1 over g1 and g9 over g9), the large bend and the twists at the junctions may also be responsible. The sugar-phosphate backbones of the 3′-end dinucleoside overhangs snuggly fit into the minor grooves of adjacent double helical stacks. The 3′-terminal deoxycytidines form antiparallel hemiprotonated trans (C·C)+ pairs with symmetry related deoxycytidines, while the penultimate adenines form base triples (a*g·c) with the capping g·c base pairs of the hexamer duplex with the adenine (a7) at one end being syn and at the other anti. These triple interactions are the same as those found in the tetrahymena ribozyme and group I intron.

Crystal structures of two plasmid copy control related RNA duplexes: An 18 base pair duplex at 1.20 A resolution and a 19 base pair duplex at 1.55 A resolution.

The structures of two RNA duplexes, whose sequences correspond to portions of the ColE1 plasmid copy control RNA I and RNA II, have been determined. Crystals containing the 18mers 5'-CA CCGUUGGUAGCGGUGC-3' and 5'-CACCGCUACCAACGGUGC-3' diffract to 1.20 A resolution while those containing the 19mers 5'-GCACCGUUGGUAGCGGUGC-3' and 5'-GCACCGCUACCAACGGUGC-3' diffract to 1.55 A resolution. Both duplexes are standard A form, with Watson-Crick base pairing throughout. Use of anisotropic atomic displacement factors in refinement of the 1.20 A structure dramatically improved refinement statistics, resulting in a final R(free) of 15.0% and a crystallographic R-factor of 11.6%. Perhaps surprisingly, these crystals of the 18 base pair RNA exhibit a 36-fold static disorder, resulting in a structure with a single sugar-phosphate backbone conformation and an averaged base composition at each residue. Since the sugar-phosphate backbone structure is identical in the 36 different nucleotides that are superimposed, there can be no sequence-dependent variation in the structure. The average ribose pucker amplitude is 45.8 degrees for the 18 base pair structure and 46.4 degrees for the 19 base pair structure; these values are respectively 19% and 20% larger than the average pucker amplitude reported from nucleoside crystal structures. A standard RNA water structure, based on analysis of the hydration of these crystal structures and that of the TAR RNA stem [Ippolito, J. A., and Steitz, T. A. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 9819-9824], has been derived, which has allowed us to predict water positions in lower resolution RNA crystal structures. We report a new RNA packing motif, in which three pro-S(p) phosphate oxygens interact with an ammonium ion.

Ribonuclease III Processing of Coaxially Stacked RNA Helices

The RNase III family of endoribonucleases participates in maturation and decay of cellular and viral transcripts by processing of double-stranded RNA. RNase III degradation is inherent to most antisense RNA-regulated gene systems in Escherichia coli. In the hok/sok system from plasmid R1, Sok antisense RNA targets the hok mRNA for RNase III-mediated degradation. An intermediate in the pairing reaction between Sok RNA and hok mRNA forms a three-way junction. A complex between a chimeric antisense RNA and hok mRNA that mimics the three-way junction was cleaved by RNase III both in vivo and in vitro. Footprinting using E117A RNase III binding to partially complementary RNAs showed protection of the 13 base pairs of interstrand duplex and of the bottom part of the transcriptional terminator hairpin of the antisense RNA. This suggests that the 13 base pairs of RNA duplex are coaxially stacked on the antisense RNA terminator stem-loop and that each stem forms a monomer half-site, allowing symmetrical binding of the RNase III dimer. This processing scheme shows an unanticipated diversity in RNase III substrates and may have a more general implication for RNA metabolism.

Placement of protein and RNA structures into a 5 A-resolution map of the 50S ribosomal subunit.

We have calculated at 5.0 A resolution an electron-density map of the large 50S ribosomal subunit from the bacterium Haloarcula marismortui by using phases derived from four heavy-atom derivatives, intercrystal density averaging and density-modification procedures. More than 300 base pairs of A-form RNA duplex have been fitted into this map, as have regions of non-A-form duplex, single-stranded segments and tetraloops. The long rods of RNA crisscrossing the subunit arise from the stacking of short, separate double helices, not all of which are A-form, and in many places proteins crosslink two or more of these rods. The polypeptide exit channel was marked by tungsten cluster compounds bound in one heavy-atom-derivatized crystal. We have determined the structure of the translation-factor-binding centre by fitting the crystal structures of the ribosomal proteins L6, L11 and L14, the sarcin-ricin loop RNA, and the RNA sequence that binds L11 into the electron density. We can position either elongation factor G or elongation factor Tu complexed with an aminoacylated transfer RNA and GTP onto the factor-binding centre in a manner that is consistent with results from biochemical and electron microscopy studies.

Melting studies of short DNA hairpins: influence of loop sequence and adjoining base pair identity on hairpin thermodynamic stability.

Spectroscopic and calorimetric melting studies of 28 DNA hairpins were performed. These hairpins form by intramolecular folding of 16 base self-complementary DNA oligomer sequences. Sequence design dictated that the hairpin structures have a six base pair duplex linked by a four base loop and that the first five base pairs in the stem are the same in every molecule. Only loop sequence and identity of the duplex base pair closing the loop vary for the set of hairpins. For these DNA samples, melting studies were carried out to investigate effects of the variables on hairpin stability. Stability of the 28 oligomers was ascertained from their temperature-induced melting transitions in buffered 115 mM Na(+) solvent, monitored by ultraviolet absorbance and differential scanning calorimetry (DSC). Experiments revealed the melting temperatures of these molecules range from 32.4 to 60.5 degrees C and are concentration independent over strand concentrations of 0.5 to 260 microM; thus, as expected for hairpins, the melting transitions are apparently unimolecular. Model independent thermodynamic transition parameters, DeltaH(cal), DeltaS(cal), and DeltaG(cal), were determined from DSC measurements. Model dependent transition parameters, DeltaH(vH), DeltaS(vH), and DeltaG(vH) were estimated from a van't Hoff (two-state) analysis of optical melting transitions. Results of these studies reveal a significant sequence dependence to DNA hairpin stability. Thermodynamic parameters evaluated by either procedure reveal the transition enthalpy, DeltaH(cal) (DeltaH(vH)) can differ by as much as 20 kcal/mol depending on sequence. Similarly, values of the transition entropy DeltaS(cal) (DeltaS(vH)) can differ by as much as 60 cal/Kmol (eu) for different molecules. Differences in free energies DeltaG(cal) (DeltaG(vH)) are as large as 4 kcal/mol for hairpins with different sequences. Comparisons between the model independent calorimetric values and the thermodynamic parameters evaluated assuming a two-state model reveal that 10 of the 28 hairpins display non-two-state melting behavior. The database of sequence-dependent melting free energies obtained for the hairpins was employed to extract a set of n-n (nearest-neighbor) sequence dependent loop parameters that were able to reproduce the input data within error (with only two exceptions). Surprisingly, this suggests that the thermodynamic stability of the DNA hairpins can in large part be reasonably represented in terms of sums of appropriate nearest-neighbor loop sequence parameters.

An A-DNA structure with two independent duplexes in the asymmetric unit.

The crystal and molecular structure of the self-complementary A-DNA decamer sequence d(G4CGC4) was solved at 1.9 A resolution. The decamer crystallizes in space group P21 with two independent duplexes in the asymmetric unit. Duplex 1 has interactions which are distributed symmetrically about its length compared with duplex 2. The two end base pairs of duplex 1 have a similar NH.O hydrogen-bond pattern involving GGC segments of duplex 2 and a symmetry-related neighbour, while the end base pairs of duplex 2 interact with the GCC and GGG segments of its symmetry-related neighbours through NH.O and NH.N hydrogen bonds and a water-mediated hydrogen bond between the carboxyl groups of C40 and C8. In addition to the C4'-C5' torsion angle gamma assuming the trans conformation in certain steps, this angle also adopts the gauche- conformation at C37 as opposed to the preferred gauche+ conformation, with a concomitant change in phosphodiester P-O5' (alpha) in the opposite sense. This facilitates stacking between adjacent bases. The study suggests that the structural alterations in the two molecules in the asymmetric unit originate from an inherent propensity of the d(G4CGC4) base sequence for varied intermolecular interactions and malleability.

Decreased imino proton exchange and base-pair opening in the IHF-DNA complex measured by NMR

Integration Host Factor, IHF, is an E. coli DNA binding protein that imposes a substantial bend on DNA. Previous footprinting studies and bending assays have characterized several recognition sequences in the bacterial and lambda phage genome as unique in the way they are bound by IHF. We have chosen one of the lambda phage sites, H1, for study because it presents a small yet sequence-specific substrate for NMR analysis of the complex. A 19 base-pair duplex, H19, corresponding to the recognition sequence at the H1 site was constructed by isotopically labeling one of the strands with 15N. (1H, 15N) heteronuclear NMR experiments aided in assigning the imino proton resonances of the DNA alone and in complex with IHF. The NMR results are consistent with a mode of binding observed in the recent crystal structure of IHF bound to another of its sites from the lambda phage genome. Additionally, the dramatic change that IHF imposes on the imino proton chemical shifts is indicative of a severe deviation from canonical B-DNA structure. In order to understand the dynamic properties of the DNA in the complex with IHF, the exchange rates of the imino protons with the solvent have been measured for H19 with and without IHF bound. A drastic reduction in exchange is observed for the imino protons in the IHF bound DNA. In the DNA-protein complex, groups of adjacent base-pair exchange at the same rate, and appear to close more slowly than the rate of imino proton exchange with bulk water, since their exchange rate is independent of catalyst concentration. We infer that segments of the double helix as large as 6 bp open in a cooperative process, and remain open much longer than is typical for opening fluctuations in naked duplex DNA. We discuss these results in terms of the specific protein-DNA contacts observed in the crystal structure.

Threading Intercalators as a New DNA Structural Probe

Abstract Nogalamycin is a threading intercalator and when it binds with double stranded DNA, one of the two sugar substituents goes between adjacent base pairs of DNA to form a stable complex in which the two substituents of nogalamycin are projecting out in each groove of DNA. Since the molecular volume of the substituents of nogalamycin is larger than that of the space created through the motion of DNA strands constituting the double helix, a cleavage of hydrogen bonds in the base pairs of DNA duplex is necessary to form such a complex. The structure of double-stranded DNA is dynamic, and the base pair cleavage (base pair opening) and re-formation (closing) occur frequently at ambient temperature. A wider variety of threading intercalators is obtainable synthetically, and they include several families such as 1,5-disubstituted anthraquinones, 9,10-disubstituted anthracenes, and naphthalene bis(dicarboximide) derivatives. Kinetic study of the complex formation and dissociation of naphthalene bis(dicarboximide) carrying substituents of different size have made one recognize the dynamic character of duplex DNA in intercalation reaction. A cyclic bis-intercalator was also able to form a threading complex of a catenane-like structure. A thorough study of the binding behavior of natural and synthetic threading intercalators with DNA revealed that a proper design of substituents on threading intercalators can produce ligands which specifically bind either to single- or double-stranded DNA. These ligands, synthetic ones in particular, will hopefully serve not only as new DNA structural probes but also as chemotherapeutic agents.

High Base Pair Opening Rates in Tracts of GC Base Pairs

Sequence-dependent structural features of the DNA double helix have a strong influence on the base pair opening dynamics. Here we report a detailed study of the kinetics of base pair breathing in tracts of GC base pairs in DNA duplexes derived from 1H NMR measurements of the imino proton exchange rates upon titration with the exchange catalyst ammonia. In the limit of infinite exchange catalyst concentration, the exchange times of the guanine imino protons of the GC tracts extrapolate to much shorter base pair lifetimes than commonly observed for isolated GC base pairs. The base pair lifetimes in the GC tracts are below 5 ms for almost all of the base pairs. The unusually rapid base pair opening dynamics of GC tracts are in striking contrast to the behavior of AT tracts, where very long base pair lifetimes are observed. The implication of these findings for the structural principles governing spontaneous helix opening as well as the DNA-binding specificity of the cytosine-5-methyltransferases, where flipping of the cytosine base has been observed, are discussed.

Effect of electrostatic interactions on the structure and dynamics of a model polyelectrolyte. II. Intermolecular correlations

The peak in the small angle x-ray scattering and the dynamic light-scattering slow mode for a 20 base-pair duplex oligonucleotide (“B-DNA”) are studied as functions of oligonucleotide and added-salt (NaCl) concentrations. Both the x-ray peak intensity and the relative intensity of the slow mode decrease as the added-salt concentration is increased. The hydrodynamic radius of the slow mode increases as the added-salt concentration is decreased. The x-ray peak gradually disappears with increasing salt while the slow mode decreases in intensity, but still has some residual intensity at the highest added-salt concentration studied. There is no abrupt change in either the peak or the slow mode with increasing salt. The existence and behavior of both the x-ray peak and the slow mode indicate local ordering in the solution due to electrostatic forces. The x-ray peak position for the oligonucleotide is correlated with the static light-scattering peak seen by other workers for dilute solutions of larger polyions. A simple model shows that the reduced electrostatic potential at the average distance between neighboring polyions is approximately the same for these polyelectrolytes. The slow mode has a hydrodynamic radius that increases with decreasing q at low added-salt concentrations, indicating a large correlation volume. The x-ray peak is a more local indicator of nearest-neighbor correlations among the polyions.

Structure and functional implications of the polymerase active site region in a complex of HIV-1 RT with a double-stranded DNA template-primer and an antibody fab fragment at 2.8 Å resolution

The structure of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) complexed with a 19-mer/18-mer double-stranded DNA template-primer (dsDNA) and the Fab fragment of monoclonal antibody 28 (Fab28) has been refined at 2.8 Å resolution. The structures of the polymerase active site and neighboring regions are described in detail and a number of novel insights into mechanisms of polymerase catalysis and drug inhibition are presented. The three catalytically essential amino acid residues (Asp110, Asp185, and Asp186) are located close to the 3′ terminus of the primer strand. Observation of a hydrogen bond between the 3′-OH of the primer terminus and the side-chain of Asp185 suggests that the carboxylate of Asp185 could act as a general base in initiating the nucleophilic attack during polymerization. Nearly all of the close protein-DNA interactions involve atoms of the sugar-phosphate backbone of the nucleic acid. However, the phenoxyl side-chain of Tyr183, which is part of the conserved YMDD motif, has hydrogen-bonding interactions with nucleotide bases of the second duplex base-pair and is predicted to have at least one hydrogen bond with all Watson-Crick base-pairs at this position. Comparison of the structure of the active site region in the HIV-1 RT/dsDNA complex with all other HIV-1 RT structures suggests that template-primer binding is accompanied by significant conformational changes of the YMDD motif that may be relevant for mechanisms of both polymerization and inhibition by non-nucleoside inhibitors. Interactions of the “primer grip” (the β12-β13 hairpin) with the 3′ terminus of the primer strand primarily involve the main-chain atoms of Met230 and Gly231 and the primer terminal phosphate. Alternative positions of the primer grip observed in different HIV-1 RT structures may be related to conformational changes that normally occur during DNA polymerization and translocation. In the vicinity of the polymerase active site, there are a number of aromatic residues that are involved in energetically favorable π-π interactions and may be involved in the transitions between different stages of the catalytic process. The protein structural elements primarily responsible for precise positioning of the template-primer (including the primer grip, template grip, and helices αH and αI of the p66 thumb) can be thought of functioning as a “translocation track” that guides the relative movement of nucleic acid and protein during polymerization.

Orienting DNA Helices on Gold Using Applied Electric Fields

Gold surfaces modified with thiol-derivatized DNA duplexes have been investigated as a function of applied electrochemical potential via atomic force microscopy (EC-AFM). At open circuit, monolayers of well-packed DNA helices form with a film depth of 45(3) Å. On the basis of the anisotropic dimensions of these 15 base pair duplexes (20 Å in diameter versus 50 Å in length), this corresponds to an average ∼45° orientation of the helical axis with respect to the gold surface. Under potential control, the monolayer thickness (and therefore the orientation of the helices) changes dramatically with applied potential. At potentials negative of ∼0.45 V (versus a Ag wire quasi-reference electrode) film thicknesses of ∼55 Å are observed, whereas at more positive potentials the monolayer thickness drops to a limiting value of ∼20 Å. These results are consistent with a morphology change in which the helices either stand straight up or lie flat down on the metal surface, depending on the electrode potential relative to the potential of zero charge (pzc). This voltage-induced morphology change is reversible and effectively constitutes a nanoscale mechanical “switch”.

The helix-coil transition of DNA duplexes and hairpins observed by multiple fluorescence parameters.

The thermal denaturation of 8-20-bp DNA duplexes labeled with fluorescein and tetramethylrhodamine at opposing 5'-ends was investigated by monitoring the fluorescence intensity of the dyes, the fluorescence anisotropy of tetramethylrhodamine, the fluorescence resonance energy transfer between fluorescein and rhodamine, and, for the 20-bp duplex, the UV absorption. Melting experiments with the single strands of the duplexes revealed that the single strands can form hairpins stabilized by only a few base pairs. The thermal denaturation curves of the duplexes were fitted well to an extended all-or-none model assuming that only the fully base-paired duplex, the maximally base-paired hairpin, and the random coil conformations are present simultaneously. The extent-of-melting versus temperature curves derived from the different spectroscopic parameters are nearly identical, provided that the analysis of the baselines is carried out correctly; the DeltaH and DeltaS of the dissociation compare well with predictions based on nearest neighbor interaction values available in the literature. Our results imply that for all the oligonucleotides other than the 34-bp oligomer, no partially melted intermediates other than hairpins are present in the reaction mixture in amounts that can be detected by our methods. The melting of the hairpins was also studied directly using single-stranded oligonucleotides. The melting of a 34-bp duplex can be accounted for by a statistical zipper model.

Rh(phen)2phi3+ as a shape-selective probe of triple helices.

RNA pur*pur-pyr and pyr*pur-pyr (pur = purine, pyr = pyrimidine) triple helices consisting of a Watson-Crick base-paired 28mer hairpin duplex and a Hoogsteen base-paired purine or pyrimidine 12mer are targeted with photoactivated cleavage by the metal complex Rh(phen)2phi3+ (phen = phenanthroline, phi = 9, 10-phenanthrenequinone diimine). The metal complex interacts with these triple helices in a structure-specific manner. Different cleavage patterns are seen with the pyr*pur-pyr and pur*pur-pyr motifs. Cleavage is seen on both of the Watson-Crick strands of the former motif and primarily on the purine Watson-Crick strand of the latter motif. Little cleavage is seen on the Hoogsteen strand for either motif. Importantly, the metal complex shows no detectable cleavage on the A-form RNA duplex in the absence of the third Hoogsteen strand. The cleavage patterns are consistent with an intercalated model for the metal complex in the triple helix. Similar cleavage is seen on DNA triple helices, but over a background of duplex cleavage. Targeting of synthetic RNA triple helices, but not duplex regions, by Rh(phen)2phi3+ provides a basis for the chemical probing of triply bonded sites in folded RNA molecules.

A Model for the Mechanism of Human Topoisomerase I

The three-dimensional structure of a 70-kilodalton amino terminally truncated form of human topoisomerase I in complex with a 22-base pair duplex oligonucleotide, determined to a resolution of 2.8 angstroms, reveals all of the structural elements of the enzyme that contact DNA. The linker region that connects the central core of the enzyme to the carboxyl-terminal domain assumes a coiled-coil configuration and protrudes away from the remainder of the enzyme. The positively charged DNA-proximal surface of the linker makes only a few contacts with the DNA downstream of the cleavage site. In combination with the crystal structures of the reconstituted human topoisomerase I before and after DNA cleavage, this information suggests which amino acid residues are involved in catalyzing phosphodiester bond breakage and religation. The structures also lead to the proposal that the topoisomerization step occurs by a mechanism termed "controlled rotation."

Bimolecular DNA triplexes: duplex extensions show implications for H-form DNA stability.

H-form DNA has recently been shown to be biologically relevant by its involvement in the process of homologous recombination [Kohwi, Y. , and Panchenko, Y. (1993) Genes Dev. 7, 1766-1778]. A bimolecular DNA triple-stranded structure (triplex) is central to the formation of H-form DNA. Understanding the formation and factors governing the stability of such bimolecular triplexes is necessary to fully elucidate the structure/function relationship of H-form DNA. In this study, we extend known information on bimolecular triplexes by examining the effect of a variable CNC base triad (where N = A, C, T, or G) on a 10 base triad triplex that mimics the triplex motif in H-form DNA. We also examine the effect that a duplex extension of four base pairs has on triplex stability and selectivity for the base N. Results from thermal denaturation experiments indicate that the fully complementary triplex is more stable than its duplex counterpart (DeltaTm = 13 degrees C) and is resistant to degradation by bovine spleen phosphodiesterase for at least 24 h at 10 degrees C. A single-base mismatch in the purine strand of the triplex structure is destabilizing (DeltaTm = approximately 20 degrees C), and all structures containing a mismatch were readily degraded by bovine spleen phosphodiesterase. An extension of four duplex base pairs onto the triplex structure affects the stability of the DNA complex and may have implications relevant to H-form DNA.