Variation of nonexchangeable proton resonance chemical shifts as a probe of aberrant base pair formation in DNA

Variation of nonexchangeable proton resonance chemical shifts for deoxycytidine and deoxy-adenosine as a function of protonation and imino tautomer formation has been determined. Protonation induces downfield shifts of proton resonances whereas formation of the rare imino tautomer induces upfield shifts. Titration curves are constructed on the basis of spectrophotometrically determined pK values. Excellent fit is obtained between theoretical titration curves and experimental data, which indicates that chemical shifts of base protons may be used to quantitatively determine the relative concentrations of either rare imino tautomeric conformations or protonated base forms. These data may be utilized as an aid in the elucidation of the nature of hydrogen bonding between mismatched base pairs in DNA oligomers containing cytosine or adenine residues. These data, in conjunction with the oligonucleotide study of Patel et al. [Patel, D. J., Kozlowski, S.A., Ikuta, S., & Itakura, K. (1984) Biochemistry 23, 3218-3226], have been used to rigorously argue the existence of a "protonated" adenine residue in the A-C mismatch. This structure allows reconciliation of the NMR solution data with crystallographic data [Hunter, W.N., Brown, T., Anand, N.N., & Kennard, O. (1986) Nature (London) 320, 552-555], which support the protonated base pair.     

Mechanism of hydroxylamine mutagenesis: tautomeric shifts and proton exchange between the promutagen N6-methoxyadenosine and cytidine

Whereas the amino, but not imino, tautomer of the promutagen N6-methoxyadenosine (OMe6A) forms planar associates (base pairs) with the potentially complementary uridine [Stolarski, R., Kierdaszuk, B., Hagberg, C.-E., & Shugar, D. (1984) Biochemistry 23, 2906-2913], it has now been found, with the aid of 1H NMR spectroscopic techniques, that only the imino tautomer of OMe6A base pairs with the potentially complementary cytidine. The association constant for such heteroassociates is more than an order of magnitude higher than that for autoassociates of OMe6A. The formation of heteroassociates is accompanied by a marked shift in tautomeric equilibrium of OMe6A, with an increase in the population of the amino form from 18% to as high as 44% and a corresponding decrease in the population of the imino species. Furthermore, the presence of cytidine in a solution of OMe6A appreciably enhances the rate of tautomeric exchange between the two tautomeric forms. Formation of planar heteroassociates between cytidine and the imino form of OMe6A is also accompanied by proton exchange between the cytidine NH2 and the N6-H of the amino form of OMe6A. The rate constants for this exchange and for tautomeric exchange, determined by the saturation transfer technique, have been measured at various concentrations and temperatures. A model is advanced for proton exchange that takes into account the interdependence of tautomeric exchange and proton exchange, as well as the role of auto- and heteroassociates. The relevance of these results to the molecular basis of hydroxylamine and methoxyamine mutagenesis and to the phenomenon of proton exchange in other systems is briefly discussed.     

Whether 2-aminopurine induces incorporation errors at the DNA replication? A quantum-mechanical answer on the actual biological issue

In this paper, we consider the mutagenic properties of the 2-aminopurine (2AP), which has intrigued molecular biologists, biophysicists and physical chemists for a long time and been widely studied by both experimentalists and theorists. We have shown for the first time using QM calculations, that 2AP very effectively produces incorporation errors binding with cytosine (C) into the wobble (w) C·2AP(w) mispair, which is supported by the N4H?N1 and N2H?N3 H-bonds and is tautomerized into the Watson–Crick (WC)-like base mispair C*·2AP(WC) (asterisk denotes the mutagenic tautomer of the base), that quite easily in the process of the thermal fluctuations acquires enzymatically competent conformation. 2AP less effectively produces transversions forming the wobble mispair with A base – A·2AP(w), stabilized by the participation of the N6H?N1 and N2H?N1 H-bonds, followed by further tautomerization A·2AP(w) → A*·2AP(WC) and subsequent conformational transition A*·2AP(WC) → A*·2AP_syn thus acquiring enzymatically competent structure. In this case, incorporation errors occur only in those case, when 2AP belongs to the incoming nucleotide. Thus, answering the question posed in the title of the article, we affirm for certain that 2AP induces incorporation errors at the DNA replication. Obtained results are consistent well with numerous experimental data.     

NMR studies of the stable mismatch purine-thymine in the self-complementary d(CGPuAATTTCG) duplex in solution

One- and two-dimensional nuclear Overhauser effect experiments demonstrate that a single hydrogen bond between a T imino proton and purine N3 is sufficient to hold the base pair dPu.dT in d(CGPuAATTTCG) by a Watson-Crick fashion rather than a Hoogsteen type. In addition, the dPu.dT base pair is well stacked with neighboring base pairs. The spin-lattice relaxation measurements at 30 and 35 degrees C of two decamers, d(CGPuAATTTCG) and d(CGAAATTTCG), reveal that the elimination of two single hydrogen bonds of dA.dT base pairs (due to the substitution of adenine for purine) in the sequence results in an increase in the overall imino proton exchange rate from 7 to 36 s-1 at the site of mismatch.     

The effect of tautomeric constant on the specificity of nucleotide incorporation during DNA replication: support for the rare tautomer hypothesis of substitution mutagenesis

The nucleoside analogue dP (6-(2-deoxy-beta-D-ribofuranosyl)-3,4-dihydro-6H,8H-pyrimido[4,5-c][1,2]oxazin-2-one) displays ambivalent hydrogen bonding characteristics whereby the imino tautomer of P can base-pair with adenine and its amino tautomer can base-pair with guanine. Fixed imino and amino tautomers of 6-methyl-3,4-dihydro-6H,8H-pyrimido[4,5-c][1,2]oxazin-2-one (N-methyl P) have been synthesised and their structures obtained by X-ray crystallography. The tautomeric constant of N-methyl P has been calculated from pK(a) values of the fixed tautomers and the kinetic parameters for the incorporation of its 5'-triphosphate (dPTP) by exonuclease-free Klenow fragment of DNA polymerase I have been determined. A strong correlation between the tautomeric constant and the incorporation specificity of dPTP is found. These results lend support to the proposal that the minor tautomeric forms of the natural bases may play an important role in substitution mutagenesis during DNA replication. Furthermore, they imply that DNA polymerases impose specific steric requirements on the base-pair during nucleotide incorporation.     

1H NMR study of the exchangeable protons of the duplex d(GCGTTGCG).d(CGCAACGC) containing a thymine photodimer.

A comparison is presented of the imino proton NMR spectra of the double stranded octamer d(GCGTTGCG).d(CGCAACGC) and the same octamer in which the two central thymine residues occur as a cis-syn thymine dimer. Except for the terminal base pairs all imino protons were detected and assigned in the NMR spectrum. The spectra show that in the thymine dimer duplex, contrary to common belief, all base pairs occur in a hydrogen bonded form, although the hydrogen bonds of the two central AT base pairs are substantially weakened. The melting temperature decreases about 13 degrees C on thymine dimer formation.     

The physicochemical essence of the purine·pyrimidine transition mismatches with Watson-Crick geometry in DNA: A·C* versa A*·C. A QM and QTAIM atomistic understanding

It was established for the first time by DFT and MP2 quantum-mechanical (QM) methods either in vacuum, so in the continuum with a low dielectric constant (ε = 4), typical for hydrophobic interfaces of specific protein-nucleic acid interactions, that the repertoire for the tautomerisation of the biologically important adenine·cytosine* (A·C*) mismatched DNA base pair, formed by the amino tautomer of the A and the imino mutagenic tautomer of the C, into the A*·C base mispair (?G = 2.72 kcal?mol^?1 obtained at the MP2 level of QM theory in the continuum with ε = 4), formed by the imino mutagenic tautomer of the A and the amino tautomer of the C, proceeds via the asynchronous concerted double proton transfer along two antiparallel H-bonds through the transition state (TS_A·C*?A*·C). The limiting stage of the A·C*→A*·C tautomerisation is the final proton transfer along the intermolecular N6H···N4 H-bond. It was found that the A·C*/A*·C DNA base mispairs with Watson–Crick geometry are associated by the N6H?N4/N4H?N6, N3H?N1/N1H?N3 and C2H?O2 H-bonds, respectively, while the TS_A·C*?A*·C is joined by the N6–H–N4 covalent bridge and the N1H?N3 and C2H?O2 H-bonds. It was revealed that the A·C*?A*·C tautomerisation is assisted by the true C2H?O2 H-bond, that in contrast to the two others conventional H-bonds exists along the entire intrinsic reaction coordinate (IRC) range herewith becoming stronger at the transition from vacuum to the continuum with ε = 4. To better understand the behavior of the intermolecular H-bonds and base mispairs along the IRC of the A·C*?A*·C tautomerisation, the profiles of their electron-topological, energetical, geometrical, polar and charge characteristics are reported in this study. It was established based on the profiles of the H-bond energies that all three H-bonds are cooperative, mutually strengthening each other. The nine key points, providing a detailed physicochemical picture of the A·C*?A*·C tautomerisation, were revealed and thoroughly examined along the IRC. It was shown that the A*·C base mispair with the population ~1 % obtained at the MP2 level of QM theory in the continuum with ε = 4 is thermodynamically and dynamically stable structure. Its lifetime was calculated to be 5.76·10^?10 s at the MP2 level of QM theory in the continuum with ε = 4. This lifetime, from the one side, enables all six low-frequency intermolecular vibrations to develop, but, from the other side, it is by order less than the time (several ns) required for the replication machinery to forcibly dissociate a base pair into the monomers during DNA replication. This means that the A*·C base mispair “slips away from the hands” of the replication machinery into the A·C* mismatched base pair. Consequently, the authors came to the conclusion that exactly the A·C* base mispair is an active player of the point mutational events and is effectively dissociated by the replication machinery into the A and C* monomers in contrast to the A*·C base mispair, playing the mediated role of a provider of the A·C* base mispair in DNA that is synthesised.     

Triplex formation by oligonucleotides containing novel deoxycytidine derivatives.

Homopurine sequences of duplex DNA are binding sites for triplex-forming oligodeoxyribopyrimidines. The interactions of synthetic duplex DNA targets with an oligodeoxyribopyrimidine containing N4-(6-amino-2-pyridinyl)deoxycytidine (1), a nucleoside designed to interact with a single C-G base pair interruption of the purine target tract, was studied by UV melting, circular dichroism spectroscopy and dimethylsulfate alkylation experiments. Nucleoside 1 supports stable triplex formation at pH 7.0 with formation of a 1-Y-Z triad, where Y-Z is a base pair in the homopurine tract of the target. Selective interaction was observed when Y-Z was C-G, although A-T and, to a lesser extent, T-A and G-C base pairs were also recognized. The circular dichroism spectra of the triplex having a 1-C-G triad were similar to those of a triplex having a C(+)-G-C triad, suggesting that the overall structures of the two triplexes are quite similar. Removal of the 6-amino group from 1 essentially eliminated triplex formation. Reaction of a triplex having the 1-C-G triad with dimethylsulfate resulted in a 50% reduction of methylation of the G residue of this triad. In contrast, the G of a similar triplex containing a U-C-G triad was not protected from methylation by dimethylsulfate. These results are consistent with a binding mode in which the 6-amino-2-pyridinyl group of 1 spans the major groove of the target duplex at the 1-C-G binding site and forms a hydrogen bond with the O6 of G. An additional stabilizing hydrogen bond could form between the N4 of the imino tautomer of 1 and the N4 amino group of C.     

Multiple structures for the 2-aminopurine-cytosine mispair

The base pair formed between 2-aminopurine (2AP) and cytosine (C) is an intermediate in transition mutations generated by 2AP. To date, several structures have been proposed for the 2AP-C mispair, including those involving a rare tautomer, a protonated base pair, and a neutral wobble structure. In this paper, we describe a series of UV, fluorescence, and NMR studies which demonstrate that an equilibrium exists between the neutral wobble and the protonated Watson-Crick structures. The apparent pK value for the transition between the structures is 5.9-6.0. Formation of a Watson-Crick base pair is accomplished predominantly by protonation of the 2AP residue as indicated by UV spectral changes, fluorescence quenching, and changes in proton chemical shifts. Rapid transfer of the shared proton between the 2AP and cytosine residues is indicated by the rapid exchange of the cytosine amino protons of the protonated Watson-Crick configuration. The relative contribution of the neutral wobble and protonated Watson-Crick configurations to 2AP-induced transition mutations is discussed.     

Solution structure of an unusually stable RNA tetraplex containing G- and U-quartet structures.

A model for the solution structure of an RNA tetraplex, (rUGGGGU)4, has been obtained by two-dimensional NMR spectroscopy and molecular dynamics. The molecule is parallel stranded and Hoogsteen base-paired in 50 mM KCl, and it is so stable that three of its six imino protons have exchange half-lives measured in days at 40 degrees C. The tetraplex is stabilized by base stacking and by the hydrogen bonds in four G quartets and at least one U quartet. This is the first indication of the existence of U-quartet structures of which we are aware.     

1H- and 13C-NMR, FTIR, UV-VIS, ESI-MS, and PM5 studies as well as emission properties of a new Schiff base of gossypol with 5-methoxytryptamine and a new hydrazone of gossypol with dansylhydrazine

A new Schiff base of gossypol with 5-methoxytryptamine (GSTR) and a new hydrazone of gossypol with dansylhydrazine (GHDH) have been synthesized and studied by Fourier transform infrared (FTIR), 1H and 13C nuclear magnetic resonance (NMR), ultraviolet-visible (UV-VIS), electrospray ionization-mass spectroscopy (ESI-MS) as well as the parametric method PM5. The spectroscopic methods have provided clear evidence that GSTR exists in chloroform solution as an enamine-enamine tautomer, whereas GHDH is present in chloroform as a N-imine-N-imine tautomer. The fluorescence spectra of both compounds indicate that their quantum yield of fluorescence is increased by one or two orders of magnitude compared to that of pure gossypol. The ESI-MS spectra of the 1:1 mixtures of GSTR or GHDH with formic acid have demonstrated that both compounds exist as protonated monomers in the gas phase, whereas GHDH can also exist in a stable protonated dimeric structure. The structures of the stable tautomers are calculated and visualized using the PM5 semiempirical method. The intra- and intermolecular hydrogen bonds within these structures are discussed.     

Probing the role of hydrogen bonds in the stability of base pairs in double-helical DNA

Aromatic stacking and hydrogen bonding between nucleobases are two of the key interactions responsible for stabilization of DNA double-helical structures. The present work aims at defining the specific contributions of these interactions to the stability of individual base pairs in DNA. The two DNA double helices investigated are formed, respectively, by the palindromic base sequences 5'-dCCAACGTTGG-3' and 5'-dCGCAGATCTGCG-3'. The strength of the N==H...N inter-base hydrogen bond in each base pair is characterized from the measurement of the protium-deuterium fractionation factor of the corresponding imino proton using NMR spectroscopy. The structural stability of each base pair is evaluated from the exchange rate of the imino proton, measured by NMR. The results reveal that the fractionation factors of the imino protons in the two DNA double helices investigated fall within a narrow range of values, between 0.92 and 1.0. In contrast, the free energies of structural stabilization for individual base pairs span 3.5 kcal/mol, from 5.2 to 8.7 kcal/mol (at 15 degrees C). These findings indicate that, in the two DNA double helices investigated, the strength of N==H...N inter-base hydrogen bonds does not change significantly depending on the nature or the sequence context of the base pair. Hence, the variations in structural stability detected by proton exchange do not involve changes in the strength of inter-base hydrogen bonds. Instead, the results suggest that the energetic identity of a base pair is determined by the number of inter-base hydrogen bonds, and by the stacking interactions with neighboring base pairs.     

Structure and dynamics of a fluorescent DNA oligomer containing the EcoRI recognition sequence: fluorescence, molecular dynamics, and NMR studies

The self-complementary DNA decamer duplex d(CTGAATTCAG)2 and its modified counterpart d(CTGA[2AP]TTCAG)2, where the innermost adenine (6-aminopurine) has been replaced with the fluorescent analogue 2-aminopurine (2AP), have been studied by fluorescence and NMR spectroscopy and simulated by molecular dynamics. Both decamers are recognized and cleaved by the EcoRI restriction endonuclease. 2D NMR results show that both decamers have a standard B-type conformation below 20 degrees C, though a disturbance exists to the 5' side of the 2AP site which may originate from increased local mobility. The fluorescence and fluorescence anisotropy decays of both decamers, as well as the one containing 2AP in only one chain, were studied as a function of temperature. The data show that the 2AP base exists in a temperature-dependent distribution of states and shows rapid motions, suggesting interconversion among these states on a time scale of about 10(-10) s. The integrated fluorescence of the decamer with 2AP in both chains shows a large increase around the helix melting temperature whereas the decamer with one 2AP shows only a mild increase, showing that the mixed helix has a different structural transition as sensed by the 2AP base. The data suggest a model of conformational states which have distinct fluorescence decay times. The various states may differ in the degree of base stacking. Fluctuations in the degree of stacking of the A or 2AP base are supported by molecular dynamics simulations, which additionally show that the 2AP-T or A-T base pair hydrogen bonds remain intact during these large motions.     

NMR studies on an oligodeoxynucleotide containing 2-aminopurine opposite adenine

A heteroduplex containing the mismatch 2-aminopurine (AP)-adenine has been synthesized and studied by proton NMR. The mismatch was incorporated into the sequence d[CGG(AP)GGC].d-(GCCACCG). One-dimensional nuclear Overhauser effect measurements in H2O and two-dimensional nuclear Overhauser effect spectra in D2O show AP.A base pairs in a wobble structure in which both bases are in the anti conformation. The adenine is stacked well in the helix, but the helix twist between the adenine and neighboring cytosine in the 3' direction is unusually small. As a result, the aminopurine on the opposite strand is somewhat pushed out of the helix. From the measurements of the imino proton line widths, the two adjacent G.C base pairs are not found to be significantly destabilized by the presence of the purine-purine wobble pair.     

Proton nuclear magnetic resonance investigation of the conformation and dynamics in the synthetic deoxyribonucleic acid decamers d(ATATCGATAT) and d(ATATGCATAT)

A variety of one-dimensional proton NMR methods have been used to investigate the properties of two synthetic DNA decamers, d(ATATCGATAT) and d(ATATGCATAT). These results, in conjunction with the results of two-dimensional NMR experiments, permit complete assignment of the base proton resonances. Low-field resonances were assigned by sequential "melting" of the A . T base pairs and by comparison of the spectra of the two decamers. Below 20 degree C spin-lattice relaxation is dominated by through-space dipolar interactions. A substantial isotope effect on the G imino proton relaxation is observed in 75% D2O, confirming the importance of the exchangeable amino protons in the relaxation process. A somewhat smaller isotope effect is observed on the T imino proton relaxation. At elevated temperatures spin-lattice relaxation of the imino protons is due to proton exchange with solvent. Apparent activation energies for exchange vary from 36 kcal/base pair for base pairs (3,8) to 64 kcal/mol for the most interior base pairs (5,6), indicating that disruption of part, or all, of the double helix contributes significantly to the exchange of the imino protons in these decamers. By contrast, single base pair opening events are the major low-temperature pathways for exchange from A X T and G X C base pairs in the more stable higher molecular weight DNA examined in other studies. The temperature dependence of the chemical shifts and line widths of certain aromatic resonances indicates that the interconversion between the helix and coil states is not in fast exchange below the melting temperature, Tm. Within experimental error, no differential melting of base pairs was found in either molecule, and both exhibited melting points Tm = 50-52 degrees C. Spin-spin and spin-lattice relaxation rates of the nonexchangeable protons (TH6, AH8, and AH2) are consistent with values calculated by using an isotropic rotor model with a rotational correlation time of 6 ns and interproton distances appropriate for B-family DNA. The faster decay of AH8 compared with GH8 is attributed to an interaction between the thymine methyl protons and the AH8 protons in adjacent adenines (5'ApT3'). The base protons (AH8, GH8, and TH6) appear to be located close (1.9-2.3 A) to sugar H2',2" protons.(ABSTRACT TRUNCATED AT 400 WORDS)     

Base pair mismatches and carcinogen-modified bases in DNA: an NMR study of G.T and G.O4meT pairing in dodecanucleotide duplexes

High-resolution two-dimensional NMR studies have been completed on the self-complementary d(C-G-C-G-A-G-C-T-T-G-C-G) duplex (designated G.T 12-mer) and the self-complementary d(C-G-C-G-A-G-C-T-O4meT-G-C-G) duplex (designated G.O4meT 12-mer) containing G.T and G.O4meT pairs at identical positions four base pairs in from either end of the duplex. The exchangeable and nonexchangeable proton resonances have been assigned from an analysis of two-dimensional nuclear Overhauser enhancement (NOESY) spectra for the G.T 12-mer and G.O4meT 12-mer duplexes in H2O and D2O solution. The guanosine and thymidine imino protons in the G.T mismatch resonate at 10.57 and 11.98 ppm, respectively, and exhibit a strong NOE between themselves and to imino protons of flanking base pairs in the G.T 12-mer duplex. These results are consistent with wobble pairing at the G.T mismatch site involving two imino proton-carbonyl hydrogen bonds as reported previously [Hare, D. R., Shapiro, L., & Patel, D. J. (1986) Biochemistry 25, 7445-7456]. In contrast, the guanosine imino proton in the G.O4meT pair resonates at 8.67 ppm. The large upfield chemical shift of this proton relative to that of the imino proton resonance of G in the G.T mismatch or in G.C base pairs indicates that hydrogen bonding to O4meT is either very weak or absent. This guanosine imino proton has an NOE to the OCH3 group of O4meT across the pair and NOEs to the imino protons of flanking base pairs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dynamics of uracil and 5-fluorouracil in DNA

The prodrug 5-fluorouracil (5-FU), after activation into 5-F-dUMP, is an extensively used anticancer agent that inhibits thymidylate synthase and leads to increases in dUTP and 5-F-dUTP levels in cells. One mechanism for 5-FU action involves DNA polymerase mediated incorporation of dUTP and 5-F-dUTP into genomic DNA leading to U/A, 5-FU/A, or 5-FU/G base pairs. These uracil-containing lesions are recognized and excised by several human uracil excision repair glycosylases (hUNG2, hSMUG2, and hTDG) leading to toxic abasic sites in DNA that may precipitate cell death. Each of these enzymes uses an extrahelical base recognition mechanism, and previous studies with UNG have shown that extrahelical recognition is facilitated by destabilized base pairs possessing kinetically enhanced base pair opening rates. Thus, the dynamic properties of base pairs containing 5-FU and U are an important unknown in understanding the role of these enzymes in damage recognition and prodrug activation. The pH dependence of the (19)F NMR chemical shift of 5-FU imbedded in a model trinucleotide was used to obtain a pK(a) = 8.1 for its imino proton (10 °C). This is about 1.5 units lower than the imino protons of uracil or thymine and indicates that at neutral pH 5-FU exists significantly as an ionized tautomer that can mispair with guanine during DNA replication. NMR imino proton exchange measurements show that U/A and 5-FU/A base pairs open with rate constants (k(op)) that are 6- and 13-fold faster than a T/A base pair in the same sequence context. In contrast, these same base pairs have apparent opening equilibrium constants (αK(op)) that differ by less than a factor of 2, indicating that the closing rates (k(cl)) are enhanced by nearly equal amounts as k(op). These dynamic measurements are consistent with the previously proposed kinetic trapping model for extrahelical recognition by UNG. In this model, the enhanced intrinsic opening rates of destabilized base pairs allow the bound glycosylase to sample dynamic extrahelical excursions of thymidine and uracil bases as the first step in recognition.     

Mechanism of inactivation of ornithine transcarbamoylase by Ndelta -(N'-Sulfodiaminophosphinyl)-L-ornithine, a true transition state analogue? Crystal structure and implications for catalytic mechanism

The crystal structure is reported at 1.8 A resolution of Escherichia coli ornithine transcarbamoylase in complex with the active derivative of phaseolotoxin from Pseudomonas syringae pv. phaseolicola, N(delta)-(N'-sulfodiaminophosphinyl)-l-ornithine. Electron density reveals that the complex is not a covalent adduct as previously thought. Kinetic data confirm that N(delta)-(N'-sulfodiaminophosphinyl)-l-ornithine exhibits reversible inhibition with a half-life in the order of approximately 22 h and a dissociation constant of K(D) = 1.6 x 10(-12) m at 37 degrees C and pH 8.0. Observed hydrogen bonding about the chiral tetrahedral phosphorus of the inhibitor is consistent only with the presence of the R enantiomer. A strong interaction is also observed between Arg(57) Nepsilon and the P-N-S bridging nitrogen indicating that imino tautomers of N(delta)-(N'-sulfodiaminophosphinyl)-l-ornithine are present in the bound state. An imino tautomer of N(delta)-(N'-sulfodiaminophosphinyl)-l-ornithine is structurally analogous to the proposed reaction transition state. Hence, we propose that N(delta)-(N'-sulfodiaminophosphinyl)-l-ornithine, with its three unique N-P bonds, represents a true transition state analogue for ornithine transcarbamoylases, consistent with the tight binding kinetics observed.     

Structure and dynamics of thioguanine-modified duplex DNA

Mercaptopurine and thioguanine, two of the most widely used antileukemic agents, exert their cytotoxic, therapeutic effects by being incorporated into DNA as deoxy-6-thioguanosine. However, the molecular mechanism(s) by which incorporation of these thiopurines into DNA translates into cytotoxicity is unknown. The solution structure of thioguanine-modified duplex DNA presented here shows that the effects of the modification on DNA structure were subtle and localized to the modified base pair. Specifically, thioguanine existed in the keto form, formed weakened Watson-Crick hydrogen bonds with cytosine and caused a modest approximately 10 degrees opening of the modified base pair toward the major groove. In contrast, thioguanine significantly altered base pair dynamics, causing an approximately 80-fold decrease in the base pair lifetime with cytosine compared with normal guanine. This perturbation was consistent with the approximately 6 degrees C decrease in DNA melting temperature of the modified oligonucleotide, the 1.13 ppm upfield shift of the thioguanine imino proton resonance, and the large increase in the exchange rate of the thioguanine imino proton with water. Our studies provide new mechanistic insight into the effects of thioguanine incorporation into DNA at the level of DNA structure and dynamics, provide explanations for the effects of thioguanine incorporation on the activity of DNA-processing enzymes, and provide a molecular basis for the specific recognition of thioguanine-substituted sites by proteins. These combined effects likely cooperate to produce the cellular responses that underlie the therapeutic effects of thiopurines.