Ionized and wobble base-pairing for bromouracil-guanine in equilibrium under physiological conditions. A nuclear magnetic resonance study on an oligonucleotide containing a bromouracil-guanine base-pair as a function of pH

A one and two-dimensional nuclear magnetic resonance study of a non-selfcomplementary oligonucleotide containing a central 5-bromouracil-guanine pair is reported. For these two bases three types of hydrogen bonding schemes could exist; wobble, rare tautomer and ionized. The two-dimensional spectra of non-exchangeable protons together with one-dimensional spectra recorded in water show that at pH 7.0 the predominant species is a right-handed B-form DNA in which the brU.G pair has wobble geometry. On raising the pH we observe a transition monitored by proton chemical shift changes for the brU.G and adjacent base-pairs. The mid-point of the transition was observed at pH 8.6. Spectra recorded at pH 9.8 show that the helix remains intact with B form conformation. It is shown that this high pH form has an ionized brU.G base-pair now in Watson-Crick geometry. Thus under physiological conditions an equilibrium exists between wobble and ionized structures.     

O6-ethylguanine carcinogenic lesions in DNA: an NMR study of O6etG.T pairing in dodecanucleotide duplexes

High-resolution two-dimensional NMR studies are reported on the self-complementary d-(C1-G2-C3-O6etG4-A5-G6-C7-T8-T9-G10-C11-G12) duplex (designated O6etG.T 12-mer) containing two symmetrically related O6etG.T lesion sites located four base pairs in from either end of the duplex. Parallel studies were undertaken on a related sequence containing O6meG.T lesion sites (designated O6meG.T 12-mer) in order to evaluate the influence of the size of the alkyl substituent on the structure of the duplex and were undertaken on a related sequence containing G.T mismatch sites (designated G.T 12-mer duplex), which served as the control duplex. The exchangeable and nonexchangeable proton and the phosphorus nuclei have been assigned from an analysis of two-dimensional nuclear Overhauser enhancement (NOE) and correlated spectra of the O6etG.T 12-mer, O6meG.T 12-mer, and G.T 12-mer duplexes in H2O and D2O solutions. The distance connectivities observed in the NOESY spectra of the O6alkG.T 12-mer duplexes establish that the helix is right-handed and all of the bases adopt an anti conformation of the glycosidic torsion angle including the O6alkG4 and T9 bases at the lesion site. The imino proton of T9 at the O6alkG.T lesion sites resonates at 8.85 ppm in the O6etG.T 12-mer duplex and at 9.47 ppm in the O6meG.T 12-mer duplex. The large upfield shift of the T9 imino proton resonance at the O6alkG4.T9 lesion site relative to that of the same proton in the G4.T9 wobble pair (11.99 ppm) and the A4.T9 Watson-Crick pair (13.95 ppm) in related sequences establishes that the hydrogen bonding of the imino proton of T9 to O6alkG4 is either very weak or absent. The imino proton of T9 develops NOEs to the CH3 protons of the O6etG and O6meG alkyl groups across the base pair, as well as to the imino and H5 protons of the flanking C3.G10 base pair and the imino and CH3 protons of the flanking A5.T8 base pair in the O6alkG.T 12-mer duplexes. These observations establish that the O6alkG4 and T9 residues are stacked into the duplex and that the O6CH3 and O6CH2CH3 groups of O6alkG4 adopt a syn orientation with respect to the N1 of the alkylated guanine.(ABSTRACT TRUNCATED AT 400 WORDS)     

Structural and dynamic properties of a fluorouracil-adenine base pair in DNA studied by proton NMR

An oligodeoxynucleotide duplex containing the chemotherapeutic agent 5-fluorouracil (FU) has been constructed by solid phase phosphotriester synthesis and has been studied in solution by proton NMR. In this study, we provide the first structural characterization of a DNA complex containing a FU.A base pair. It has been determined that the 7-mer duplex containing a central FU.A base pair adopts a normal right-handed configuration and the A residue in the FU.A pair is oriented in the normal anticonfiguration giving a Watson-Crick base pair. The significant difference between T.A and FU.A base pairs is dynamic, not structural: the FU.A base pair opens faster than normal base pairs in the oligonucleotide studied. We provide evidence that the FU.A base pair has a significantly enhanced opening rate resulting form decreased stacking of the 5-fluorouracil residue and not from the enhanced acidity of the 5'-fluorouracil imino proton.     

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)     

Equilibrium between a wobble and ionized base pair formed between fluorouracil and guanine in DNA as studied by proton and fluorine NMR

A synthetic oligonucleotide duplex containing the chemotherapeutic and mutagenic agent 5-fluorouracil paired with guanine has been studied in solution by proton and fluorine NMR. The 7-mer duplex containing a central FU.G base pair adopts a normal right-handed configuration. At low pH, the predominant base-paired structure is wobble, whereas at higher pH an ionized structure in Watson-Crick geometry is observed. The two structures are in a pH-dependent equilibrium with one another with an apparent pK of 8.3 at 23 degrees C. This is the first demonstration of an equilibrium between two distinct base pairing schemes and the first demonstration of a negatively charged base pair in DNA.     

Solution structure of an oncogenic DNA duplex containing a G.A mismatch

The DNA duplex 5'-d(GCCACAAGCTC).d(GAGCTGGTGGC), which contains a central G.A mismatch has been studied by one and two-dimensional NMR techniques. The duplex corresponds to the sequence 29-39 of the K-ras gene. The mismatch position is that of the first base of the Gly12 codon, a hot spot for mutations. The observed NOEs of the nonexchangeable protons show that both of the bases of the mismatched pair are intrahelical over a wide range of pH. However, the structure of the G.A mispair and the conformation of the central part of the duplex change with pH. This structural change shows a pK of 6.0. At low pH, the G.A bases are base paired with hydrogen bonds between the keto group of the G residue and the amino group of the A residue and, secondly, between the N7 of the G and a proton on N1 of A. This causes the G residue to adopt a syn conformation. On raising the pH, the N1-H proton of the protonated A residue is removed, and the base pair rearranges. In the neutral G.A base pair both residues adopt an anti conformation, and the mismatch is stabilized by hydrogen bonds. Our results on the exchangeable and A(H2) protons of the mismatched pair indicate a shift from a classical face-to-face two hydrogen-bonded structure to a slipped structure stabilized by bifurcated hydrogen bonds. This may be a particular characteristics of this oncogenic sequence in which the G.A error is poorly repaired.     

Extension of the range of DNA sequences available for triple helix formation: stabilization of mismatched triplexes by acridine-containing oligonucleotides.

Triple helix formation usually requires an oligopyrimidine*oligopurine sequence in the target DNA. A triple helix is destabilized when the oligopyrimidine*oligopurine target contains one (or two) purine*pyrimidine base pair inversion(s). Such an imperfect target sequence can be recognized by a third strand oligonucleotide containing an internally incorporated acridine intercalator facing the inverted purine*pyrimidine base pair(s). The loss of triplex stability due to the mismatch is partially overcome. The stability of triplexes formed at perfect and imperfect target sequences was investigated by UV thermal denaturation experiments. The stabilization provided by an internally incorporated acridine third strand oligonucleotide depends on the sequences flanking the inverted base pair. For triplexes containing a single mismatch the highest stabilization is observed for an acridine or a propanediol tethered to an acridine on its 3'-side facing an inverted A*T base pair and for a cytosine with an acridine incorporated to its 3'-side or a guanine with an acridine at its 5'-side facing an inverted G*C base pair. Fluorescence studies provided evidence that the acridine was intercalated into the triplex. The target sequences containing a double base pair inversion which form very unstable triplexes can still be recognized by oligonucleotides provided they contain an appropriately incorporated acridine facing the double mismatch sites. Selectivity for an A*T base pair inversion was observed with an oligonucleotide containing an acridine incorporated at the mismatched site when this site is flanked by two T*A*T base triplets. These results show that the range of DNA base sequences available for triplex formation can be extended by using oligonucleotide intercalator conjugates.     

NMR studies of chromomycin A3 interaction with DNA

The binding of chromomycin A3 to calf thymus DNA and poly(dG-dC) has been studied by 13C and 1H NMR with emphasis on the mode of binding, the role of Mg2+, and pH effects. The most prominent changes in the DNA base pair 13C NMR resonances upon complexation with chromomycin were observed for G and C bases, consistent with the G-C preference exhibited by this compound. Comparison of the 13C spectrum of DNA-bound chromomycin A3 with that of DNA-bound actinomycin D, a known intercalator, showed many similarities in the base pair resonances. This suggested the possibility that chromomycin A3 binds via an intercalative mechanism. 1H NMR studies in the imino proton, low-field region of the spectrum provided additional evidence in support of this binding mode. In the low-field spectrum of chromomycin A3 bound to calf thymus DNA, a small shoulder was observed on the upfield side of the G-C imino proton peak. Similarly, in the chromomycin A3 complex with poly(dG-dC), a well-resolved peak was found upfield from the G-C imino proton peak. These results are expected for ligands that bind by intercalation. Furthermore, in both the calf thymus and poly(dG-dC) drug complexes (in the presence of Mg2+) a broad peak was also present downfield (approximately 16 ppm from TSP) from the DNA imino protons. This was attributed to the C-9 phenolic hydroxyl proton on the chromomycin chromophore. Visible absorbance spectra at different pH values showed that the role of Mg2+ in the binding of chromomycin A3 to DNA is more than simple neutralization of the drug's anionic change.     

The stability of oligodeoxyribonucleotide duplexes containing degenerate bases.

Oligodeoxyribonucleotides containing N4-methoxycytosine (mo4C), N4-methoxy-5-methylcytosine (mo4m5C) and other base-analogues were synthesised and used to compare the stabilities of duplexes containing mo4C.A and mo4C.G base pairs with those containing normal and mismatch pairs. The Tm values and other thermodynamic parameters are recorded. The otherwise identical duplexes containing a mo4C.A and a mo4C.G base pair have closely similar stabilities to each other and to the corresponding duplexes containing normal base pairs, considerably greater than the stabilities of those containing mismatch pairs. Corresponding observations are recorded in dot-blot experiments using M13 cloned DNA carrying an insert complementary to the oligonucleotides; approximate Td values are given.     

T4-DNA ligase: substrate properties of synthetic DNA-duplexes with structural anomalies

Structural defects, affecting T4 DNA ligase function, were revealed with the help of synthetic DNA duplexes, containing modifications at single nick. Changes of configuration at C2' and C3' atoms of furanose in the acceptor terminus lead to total blocking of the nick sealing activity of T4 DNA ligase. On the contrary, substitution of 3'-terminal deoxyribonucleotide for ribonucleotide doesn't affect the enzyme's action. The duplex looses all of it's substrate activity if the next from the nick G.C pair is substituted for the noncomplementary G.C pair. In DNA duplexes containing an unpaired base in the nick, elimination of the extrahelical nucleotide proceeds the ligation step. In these cases the duplex substrate activity decreases depending on the extent of extrahelical base stacking into the double stranded DNA.     

Acid-induced exchange of the imino proton in G.C pairs.

Acid-induced catalysis of imino proton exchange in G.C pairs of DNA duplexes is surprisingly fast, being nearly as fast as for the isolated nucleoside, despite base-pair dissociation constants in the range of 10(-5) at neutral or basic pH. It is also observed in terminal G.C pairs of duplexes and in base pairs of drug-DNA complexes. We have measured imino proton exchange in deoxyguanosine and in the duplex (ATATAGATCTATAT) as a function of pH. We show that acid-induced exchange can be assigned to proton transfer from N7-protonated guanosine to cytidine in the open state of the pair. This is faster than transfer from neutral guanosine (the process of intrinsic catalysis previously characterized at neutral ph) due to the lower imino proton pK of the protonated form, 7.2 instead of 9.4. Other interpretations are excluded by a study of exchange catalysis by formiate and cytidine as exchange catalysts. The cross-over pH between the regimes of pH-independent and acid-induced exchange rates is more basic in the case of base pairs than in the mononucleoside, suggestive of an increase by one to two decades in the dissociation constant of the base pair upon N7 protonation of G. Acid-induced catalysis is much weaker in A.T base pairs, as expected in view of the low pK for protonation of thymidine.     

Dynamic opening of DNA during the enzymatic search for a damaged base

Uracil DNA glycosylase (UDG) removes uracil from U.A or U.G base pairs in genomic DNA by extruding the aberrant uracil from the DNA base stack. A question in enzymatic DNA repair is whether UDG and related glycosylases also use an extrahelical recognition mechanism to inspect the integrity of undamaged base pairs. Using NMR imino proton exchange measurements we find that UDG substantially increases the equilibrium constant for opening of T-A base pairs by almost two orders of magnitude relative to free B-DNA. This increase is brought about by enzymatic stabilization of an open state of the base pair without increasing the rate constant for spontaneous base pair opening. These findings indicate a passive search mechanism in which UDG uses the spontaneous opening dynamics of DNA to inspect normal base pairs in a rapid genome-wide search for uracil in DNA.     

Nearest-neighbor thermodynamics and NMR of DNA sequences with internal A.A, C.C, G.G, and T.T mismatches

Thermodynamic measurements are reported for 51 DNA duplexes with A.A, C.C, G.G, and T.T single mismatches in all possible Watson-Crick contexts. These measurements were used to test the applicability of the nearest-neighbor model and to calculate the 16 unique nearest-neighbor parameters for the 4 single like with like base mismatches next to a Watson-Crick pair. The observed trend in stabilities of mismatches at 37 degrees C is G.G > T.T approximately A.A > C.C. The observed stability trend for the closing Watson-Crick pair on the 5' side of the mismatch is G.C >/= C.G >/= A.T >/= T.A. The mismatch contribution to duplex stability ranges from -2.22 kcal/mol for GGC.GGC to +2.66 kcal/mol for ACT.ACT. The mismatch nearest-neighbor parameters predict the measured thermodynamics with average deviations of DeltaG degrees 37 = 3.3%, DeltaH degrees = 7. 4%, DeltaS degrees = 8.1%, and TM = 1.1 degrees C. The imino proton region of 1-D NMR spectra shows that G.G and T.T mismatches form hydrogen-bonded structures that vary depending on the Watson-Crick context. The data reported here combined with our previous work provide for the first time a complete set of thermodynamic parameters for molecular recognition of DNA by DNA with or without single internal mismatches. The results are useful for primer design and understanding the mechanism of triplet repeat diseases.     

The effect of cross-links on the conformational dynamics of duplex DNA.

The base pair lifetimes and apparent dissociation constants of a 21 base DNA hairpin and an analog possessing a disulfide cross-link bridging the 3'- and 5'-terminal bases were determined by measuring imino proton exchange rates as a function of exchange catalyst concentration and temperature. A comparison of the lifetimes and apparent dissociation constants for corresponding base pairs of the two hairpins indicates that the cross-link neither increases the number of base pairs involved in fraying nor alters the lifetime, dissociation constant, or the opened structure from which exchange occurs for the base pairs that are not frayed. The cross-link does, however, stabilize the frayed penultimate base pair of the stem duplex. Significantly, it appears that the disulfide cross-link is more effective at preventing fraying of the penultimate base pair than is the 5 base hairpin loop. Because this disulfide cross-link can be incorporated site specifically, and does not adversely affect static or dynamic properties of DNA, it should prove very useful in studies of nucleic acid structure and function.     

In vitro ligation of oligodeoxynucleotides containing C8-oxidized purine lesions using bacteriophage T4 DNA ligase

Ligases conduct the final stage of repair of DNA damage by sealing a single-stranded nick after excision of damaged nucleotides and reinsertion of correct nucleotides. Depending upon the circumstances and the success of the repair process, lesions may remain at the ligation site, either in the template or at the oligomer termini to be joined. Ligation experiments using bacteriophage T4 DNA ligase were carried out with purine lesions in four positions surrounding the nick site in a total of 96 different duplexes. The oxidized lesion 8-oxo-7,8-dihydroguanosine (OG) showed, as expected, that the enzyme is most sensitive to lesions on the 3' end of the nick compared to the 5' end and to lesions located in the intact template strand. In general, substrates containing the OG.A mismatch were more readily ligated than those with the OG.C mismatch. Ligations of duplexes containing the OA.T base pair (OA = 8-oxo-7,8-dihydroadenosine) that could adopt an anti-anti conformation proceeded with high efficiencies. An OI.A mismatch-containing duplex (OI = 8-oxo-7,8-dihydroinosine) behaved like OG.A. Due to its low reduction potential, OG is readily oxidized to secondary oxidation products, such as the guanidinohydantoin (Gh) and spiroiminodihydantoin (Sp) nucleosides; these lesions also contain an oxo group at the original C8 position of the purine. Ligation of oligomers containing Gh and Sp occurred when opposite A and G, although the overall ligation efficiencies were much lower than those of most OG base pairs. Steady-state kinetic studies were carried out for representative examples of lesions in the template. Km increased by 90-100-fold for OG.C-, OI.C-, OI.A-, and OA.T-containing duplexes compared to that of a G.C-containing duplex. Substrates containing Gh.A, Gh.G, Sp.A, and Sp.G base pairs exhibited Km values 20-70-fold higher than that of the substrate containing a G.C base pair, while the Km value for OG.A was 5 times lower than that for G.C.     

Crystallographic characterization of an exocyclic DNA adduct: 3,N4-etheno-2'-deoxycytidine in the dodecamer 5'-CGCGAATTepsilonCGCG-3'

Exocyclic DNA adducts are formed from metabolites of chemical carcinogens and have also been detected as endogenous lesions in human DNA. The exocyclic adduct 3,N(4)-etheno-2'-deoxycytidine (epsilon dC), positioned opposite deoxyguanosine in the B-form duplex of the dodecanucleotide d(CGCGAATTepsilonCGCG), has been crystallographically characterized at 1.8A resolution. This self-complementary oligomer crystallizes in space group P3(2)12, containing a single strand in the asymmetric unit. The crystal structure was solved by isomorphous replacement with the corresponding unmodified dodecamer structure. Exposure of both structures to identical crystal packing forces allows a detailed investigation of the influence of the exocyclic base adduct on the overall helical structure and local geometry. Structural changes are limited to the epsilon C:G and adjacent T:A and G:C base-pairs. The standard Watson-Crick base-pairing scheme, retained in the T:A and G:C base-pairs, is blocked by the etheno bridge in the epsilon C:G pair. In its place, a hydrogen bond involving O2 of epsilon C and N1 of G is present. Comparison with an epsilon dC-containing NMR structure confirms the general conformation reported for epsilon C:G, including the hydrogen bonding features. Superposition with the crystal structure of a DNA duplex containing a T:G wobble pair shows similar structural changes imposed by both mismatches. Evaluation of the hydration shell of the duplex with bond valence calculations reveals two sodium ions in the crystal.     

Structure of a G.T.A triplet in an intramolecular DNA triplex.

A 32-base DNA oligonucleotide has been studied by one- and two-dimensional 1H NMR spectroscopy and is shown to form a stable, pyr.pur.pyr, intramolecular triple helical structure, with a four C loop and a TATA loop connecting the Watson-Crick- and Hoogsteen-paired strands, respectively. This triplex contains five T.A.T base triplets, two C+.G.C base triplets, and an unusual G.T.A base triplet which disrupts the pyr.pur.pyr motif. The G.T.A triplet consists of a Watson-Crick T.A base pair, with the T situated in the "purine strand" and the A situated in the "pyrimidine strand" and a G situated in the Hoogsteen-base-paired "pyrimidine strand" hydrogen bonded to the T. The base-pairing structure of the G.T.A triplet has been investigated and has been found to involve a single hydrogen bond from the guanine amino group to the O4 carbonyl of the thymine, leaving the guanine imino proton free. The specific amino proton involved in the hydrogen bond is the H2(2) proton. This orients the guanine such that its sugar is near the thymine methyl group. The guanine sugar adopts an N-type (C3'-endo) sugar pucker in this triplet. The stability of the G.T.A triplet within pyr.pur.pyr triplexes is discussed.     

Structure of the 2-aminopurine-cytosine base pair formed in the polymerase active site of the RB69 Y567A-DNA polymerase

The adenine base analogue 2-aminopurine (2AP) is a potent base substitution mutagen in prokaryotes because of its enhanceed ability to form a mutagenic base pair with an incoming dCTP. Despite more than 50 years of research, the structure of the 2AP-C base pair remains unclear. We report the structure of the 2AP-dCTP base pair formed within the polymerase active site of the RB69 Y567A-DNA polymerase. A modified wobble 2AP-C base pair was detected with one H-bond between N1 of 2AP and a proton from the C4 amino group of cytosine and an apparent bifurcated H-bond between a proton on the 2-amino group of 2-aminopurine and the ring N3 and O2 atoms of cytosine. Interestingly, a primer-terminal region rich in AT base pairs, compared to GC base pairs, facilitated dCTP binding opposite template 2AP. We propose that the increased flexibility of the nucleotide binding pocket formed in the Y567A-DNA polymerase and increased "breathing" at the primer-terminal junction of A+T-rich DNA facilitate dCTP binding opposite template 2AP. Thus, interactions between DNA polymerase residues with a dynamic primer-terminal junction play a role in determining base selectivity within the polymerase active site of RB69 DNA polymerase.     

The stability of duplexes involving AT and/or G4EtC base pairs is not dependent on their AT/G4EtC ratio content. Implication for DNA sequencing by hybridization.

Sequencing by the recently reported hybridization technique requires the formation of DNA duplexes with similar stabilities. In this paper we describe a new strategy to obtain DNA duplexes with a thermal stability independent of their AT/GC ratio content. Melting data were acquired on 35 natural and 27 modified duplexes of a given length and of varying base compositions. Duplexes built with AT and/or G4EtC base pairs exhibit a thermal stability restrained to a lower range of temperature than that of the corresponding natural compounds (16 instead of 51 degrees C). The 16 degrees C difference in thermal stability observed between the least stable and the most stable duplex built with AT and/or G4EtC base pairs is mainly due to the sequence effect and not to their AT/G4EtC ratio content. Thus N -4-ethyl-2'-deoxycytidine (d4EtC) hybridizes specifically with natural deoxyguanosine leading to a G4EtC base pair whose stability is very close to that of the natural AT base pair. Oligonucleotide probes involving d4EtC can be easily prepared by chemical synthesis with phosphoramidite chemistry. Modified DNA targets were successfully amplified by random priming or PCR techniques using d4EtCTP, dATP, dGTP and dTTP in the presence of DNA polymerase. This new system might be very useful for DNA sequencing by hybridization.