NMR studies of the exocyclic 1,N6-ethenodeoxyadenosine adduct (epsilon dA) opposite thymidine in a DNA duplex. Nonplanar alignment of epsilon dA(anti) and dT(anti) at the lesion site

Two-dimensional proton NMR studies are reported on the complementary d(C-A-T-G-T-G-T-A-C).d(G-T-A-C-epsilon A-C-A-T-G) nonanucleotide duplex (designated epsilon dA.dT 9-mer duplex) containing 1,N6-ethenodeoxyadenosine (epsilon dA), a carcinogen-DNA adduct, positioned opposite thymidine in the center of the helix. Our NMR studies have focused on the conformation of the epsilon dA.dT 9-mer duplex at neutral pH with emphasis on defining the alignment at the dT5.epsilon dA14 lesion site. The through-space NOE distance connectivities establish that both dT5 and epsilon dA14 adopt anti glycosidic torsion angles, are directed into the interior of the helix, and stack with flanking Watson-Crick dG4.dC15 and dG6.dC13 pairs. Furthermore, the d(G4-T5-G6).d(C13-epsilon A14-C15) trinucleotide segment centered about the dT5.epsilon dA14 lesion site adopts a right-handed helical conformation in solution. Energy minimization computations were undertaken starting from six different alignments of dT5(anti) and epsilon dA14(anti) at the lesion site and were guided by distance constraints defined by lower and upper bounds estimated from NOESY data sets on the epsilon dA.dT 9-mer duplex. Two families of energy-minimized structures were identified with the dT5 displaced toward either the flanking dG4.dC15 or the dG6.dC13 base pair. These structures can be differentiated on the basis of the observed NOEs from the imino proton of dT5 to the imino proton of dG4 but not dG6 and to the amino protons of dC15 but not dC13 that were not included in the constraints data set used in energy minimization. Our NMR data are consistent with a nonplanar alignment of epsilon dA14(anti) and dT5(anti) with dT5 displaced toward the flanking dG4.dC15 base pair within the d(G4-T5-G6).d(C13-epsilon A14-C15) segment of the epsilon dA.dT 9-mer duplex.     

Solution structure of the (+)-cis-anti-benzo[a]pyrene-dA ([BP]dA) adduct opposite dT in a DNA duplex.

Minor adducts, derived from the covalent binding of anti-benzo[a]pyrene-7,8-dihydroxy-9,10-epoxide to cellular DNA, may play an important role in generating mutations and initiating cancer. We have applied a combined NMR-computational approach including intensity based refinement to determine the solution structure of the minor (+)-cis-anti-[BP]dA adduct positioned opposite dT in the d(C1-T2-C3-T4-C5-[BP]A6-C7-T8-T9-C10-C11). (d(G12-G13-A14-A15-G16-T17-G18-A19-G20+ ++-A21-G22) 11-mer duplex. The BP ring system is intercalated toward the 5'-side of the [BP]dA6 lesion site without disrupting the flanking Watson-Crick dC5.dG18 and [BP]dA6.dT17 base pairs. This structure of the (+)-cis-anti-[BP]dA.dT 11-mer duplex, containing a bay region benzo[a]pyrenyl [BP]dA adduct, is compared with the corresponding structure of the (+)-trans-anti-[BPh]dA.dT 11-mer duplex (Cosman et al., Biochemistry 32, 12488-12497, 1993), which contains a fjord region benzo[c]phenanthrenyl [BPh]dA adduct with the same R stereochemistry at the linkage site. The carcinogen intercalates toward the 5'-direction of the modified strand in both duplexes (the adduct is embedded within the same sequence context) with the buckling of the Watson-Crick [BP]dA6.dT17 base pair more pronounced in the (+)-cis-anti-[BP]dA.dT 11-mer duplex compared to its Watson-Crick [BPh]dA.dT17 base pair in the (+)-trans-anti-[BPh]dA.dT 11-mer duplex. The available structural studies of covalent polycyclic aromatic hydrocarbon (PAH) carcinogen-DNA adducts point toward the emergence of a general theme where distinct alignments are adopted by PAH adducts covalently linked to the N(6) of adenine when compared to the N(2) of guanine in DNA duplexes. The [BPh]dA and [BP]dA N(6)-adenine adducts intercalate their polycyclic aromatic rings into the helix without disruption of their modified base pairs. This may reflect the potential flexibility associated with the positioning of the covalent tether and the benzylic ring of the carcinogen in the sterically spacious major groove. By contrast, such an intercalation without modified base pair disruption option appears not to be available to [BP]dG N(2)-guanine adducts where the covalent tether and the benzylic ring are positioned in the more sterically crowded minor groove. In the case of [BP]dG adducts, the benzopyrenyl ring is either positioned in the minor groove without base pair disruption, or if intercalated into the helix, requires disruption of the modified base pair and displacement of the bases out of the helix.     

Conformational transitions in cytidine bulge-containing deoxytridecanucleotide duplexes: extra cytidine equilibrates between looped out (low temperature) and stacked (elevated temperature) conformations in solution

High-resolution homonuclear and heteronuclear two-dimensional NMR studies have been carried out on the self-complementary d(C-C-G-C-G-A-A-T-T-C-C-G-G) duplex (designated GCG 13-mer) in aqueous solution. This sequence contains an extra cytidine located between residues G3 and G4 on each strand of the duplex. The exchangeable and nonexchangeable proton resonances have been assigned from an analysis of two-dimensional nuclear Overhauser enhancement (NOESY) and correlated (COSY and relay COSY) spectra for the GCG 13-mer duplex in H2O and D2O solution. The extra cytidine at the bulge site (designated CX) results in more pronounced changes in the NOE distance connectivities for the G3-CX-G4 segment centered about the CX residue compared to the C9-C10 segment on the partner strand opposite the CX residue for the GCG 13-mer duplex at 25 degrees C. The cross-peak intensities in the short mixing time NOESY spectrum also establish that all glycosidic torsion angles including that of CX are anti in the GCG 13-mer duplex at 25 degrees C. The observed chemical shift changes for the CX base protons and the G3pCX phosphorus resonance with temperature between 0 and 40 degrees C demonstrate a temperature-dependent conformational equilibrium in the premelting transition region. The NOE and chemical shift parameters establish that the predominant conformation at low temperature (0 degree C) has the extra cytidine looped out of the helix with the flanking G3.C10 and G4.C9 base pairs stacked on each other. These results support conclusions based on earlier one-dimensional NMR studies of extra cytidine containing complementary duplexes in aqueous solution [Morden, K. M., Chu, Y. G., Martin, F. H., & Tinoco, I., Jr. (1983) Biochemistry 22, 5557-5563. Woodson, S. A., & Crothers, D. M. (1987) Biochemistry 26, 904-912]. By contrast, the chemical shift and NOE parameters demonstrate that the conformational equilibrium shifts toward a structure with a stacked extra cytidine on raising the temperature to 40 degrees C prior to the helix-coil melting transition. The most downfield shifted phosphorus resonance in the GCG 13-mer duplex has been assigned to the phosphate in the C2-G3 step, and this observation demonstrates that the perturbation in the phosphodiester backbone extends to regions removed from the (G3-CX-G4).(C9-C10) bulge site.     

Crystallization of a DNA duplex 15-mer containing unpaired bases: d(CGCGAAATTTACGCG)

Crystals of an almost self-complementary DNA 15-mer d(CGCGAAATTTACGCG) have been grown by the vapor diffusion technique at 4 degrees C. The space group is I222 with a = 37.3 A, b = 54.6 A and c = 104.8 A. Solution studies showed that the 15-mer forms a duplex with the extra adenine residue unpaired: (sequence; see text) Crystals are stable at 4 degrees C and are suitable for medium-resolution structural studies.     

Solution structure of the N-(deoxyguanosin-8-yl)-1-aminopyrene ([AP]dG) adduct opposite dA in a DNA duplex.

Solution structural studies have been undertaken on the aminopyrene-C(8)-dG ([AP]dG) adduct in the d(C5-[AP]G6-C7). d(G16-A17-G18) sequence context in an 11-mer duplex with dA opposite [AP]dG, using proton-proton distance and intensity restraints derived from NMR data in combination with distance-restrained molecular mechanics and intensity-restrained relaxation matrix refinement calculations. The exchangeable and nonexchangeable protons of the aminopyrene and the nucleic acid were assigned following analysis of two-dimensional NMR data sets on the [AP]dG.dA 11-mer duplex in H2O and D2O solution. The broadening of several resonances within the d(G16-A17-G18) segment positioned opposite the [AP]dG6 lesion site resulted in weaker NOEs, involving these protons in the adduct duplex. Both proton and carbon NMR data are consistent with a syn glycosidic torsion angle for the [AP]dG6 residue in the adduct duplex. The aminopyrene ring of [AP]dG6 is intercalated into the DNA helix between intact Watson-Crick dC5.dG18 and dC7.dG16 base pairs and is in contact with dC5, dC7, dG16, dA17, and dG18 residues that form a hydrophobic pocket around it. The intercalated AP ring of [AP]dG6 stacks over the purine ring of dG16 and, to a lesser extent dG18, while the looped out deoxyguanosine ring of [AP]dG6 stacks over dC5 in the solution structure of the adduct duplex. The dA17 base opposite the adduct site is not looped out of the helix but rather participates in an in-plane platform with adjacent dG18 in some of the refined structures of the adduct duplex. The solution structures are quite different for the [AP]dG.dA 11-mer duplex containing the larger aminopyrene ring (reported in this study) relative to the previously published [AF]dG.dA 11-mer duplex containing the smaller aminofluorene ring (Norman et al., Biochemistry 28, 7462-7476, 1989) in the same sequence context. Both the modified syn guanine and the dA positioned opposite it are stacked into the helix with the aminofluorene chromophore displaced into the minor groove in the latter adduct duplex. By contrast, the aminopyrenyl ring participates in an intercalated base-displaced structure in the present study of the [AP]dG.dA 11-mer duplex and in a previously published study of the [AP]dG.dC 11-mer duplex (Mao et al., Biochemistry 35, 12659-12670, 1996). Such intercalated base-displaced structures without hydrogen bonding between the [AP]dG adduct and dC or mismatched dA residues positioned opposite it, if present at a replication fork, may cause polymerase stalling and formation of a slipped intermediate that could produce frameshift mutations, the most dominant mutagenic consequence of the [AP]dG lesion.     

Conformation and dynamics of the Pribnow box region of the self-complementary d(C-G-A-T-T-A-T-A-A-T-C-G) duplex in solution

Nuclear magnetic resonance (NMR) has been used to monitor the conformation and dynamics of the d(C1-G2-A3-T4-T5-A6-T6-A5-A4-T3-C2-G1) self-complementary dodecanucleotide duplex (henceforth called Pribnow 12-mer), which contains a TATAAT Pribnow box and a central core of eight dA X dT base pairs. The exchangeable imino and nonexchangeable base protons have been assigned from one-dimensional intra and inter base pair nuclear Overhauser effect (NOE) measurements. Premelting conformational changes are observed at all the dA X dT base pairs in the central octanucleotide core in the Pribnow 12-mer duplex with the duplex to strand transition occurring at 55 degrees C in 0.1 M phosphate solution. The magnitude of the NOE measurements between minor groove H-2 protons of adjacent adenosines demonstrates that the base pairs are propeller twisted with the same handedness as observed in the crystalline state. The thymidine imino proton hydrogen exchange at the dA X dT base pairs has been measured from saturation recovery measurements as a function of temperature. The exchange rates and activation barriers show small variations among the four different dA X dT base pairs in the Pribnow 12-mer duplex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural studies of the O6meG.T interaction in the d(C-G-T-G-A-A-T-T-C-O6meG-C-G) duplex

High-resolution proton and phosphorus NMR studies are reported on the self-complementary d(C1-G2-T3-G4-A5-A6-T7-T8-C9-O6meG10-C11-G12) duplex (henceforth called O6meG.T 12-mer), which contains T3.O6meG10 interactions in the interior of the helix. The imino proton of T3 is observed at 9.0 ppm, exhibits a temperature-independent chemical shift in the premelting transition range, and broadens out at the same temperature as the imino proton of the adjacent G2.C11 toward the end of the helix at pH 6.8. We observed inter base pair nuclear Overhauser effects (NOEs) between the base protons at the T3.O6meG10 modification site and the protons of flanking G2.C11 and G4.C9 base pairs, indicative of the stacking of the T3 and O6meG10 bases into the helix. Two-dimensional correlated (COSY) and nuclear Overhauser effect (NOESY) studies have permitted assignment of the base and sugar H1', H2', and H2' nonexchangeable protons in the O6meG.T 12-mer duplex. The observed NOEs demonstrate an anti conformation about all the glycosidic bonds, and their directionality supports formation of a right-handed helix in solution. The observed NOEs between the T3.O6meG10 interaction and the adjacent G2.C11 and G4.C9 base pairs at the modification site exhibit small departures from patterns for a regular helix in the O6.meG.T 12-mer duplex. The phosphorus resonances exhibit a 0.5 ppm spectral dispersion indicative of an unperturbed phosphodiester backbone for the O6meG.T 12-mer duplex. We propose a model for pairing of T3 and O6meG10 at the modification site in the O6meG.T 12-mer duplex.(ABSTRACT TRUNCATED AT 250 WORDS)     

NMR studies of the exocyclic 1,N6-ethenodeoxyadenosine adduct (epsilon dA) opposite deoxyguanosine in a DNA duplex. Epsilon dA(syn).dG(anti) pairing at the lesion site

Proton NMR studies are reported on the complementary d(C-A-T-G-G-G-T-A-C).d(G-T-A-C-epsilon A-C-A-T-G) nonanucleotide duplex (designated epsilon dA.dG 9-mer duplex), which contains exocyclic adduct 1,N6-ethenodeoxyadenosine positioned opposite deoxyguanosine in the center of the helix. The present study focuses on the alignment of dG5 and epsilon dA14 at the lesion site in the epsilon dA.dG 9-mer duplex at neutral pH. This alignment has been characterized by monitoring the NOEs originating from the NH1 proton of dG5 and the H2, H5, and H7/H8 protons of epsilon dA14 in the central d(G4-G5-G6).d(C13-epsilon A14-C15) trinucleotide segment of the epsilon dA.dG 9-mer duplex. These NOE patterns establish that epsilon dA14 adopts a syn glycosidic torsion angle that positions the exocyclic ring toward the major groove edge while all the other bases including dG5 adopt anti glycosidic torsion angles. We detect a set of intra- and interstrand NOEs between protons (exchangeable and nonexchangeable) on adjacent residues in the d(G4-G5-G6).d(C13-epsilon A14-C15) trinucleotide segment which establish formation of right-handed helical conformations on both strands and stacking of the dG5(anti).epsilon dA14(syn) pair between stable dG4.dC15 and dG6.dC13 pairs. The energy-minimized conformation of the central d(G4-G5-G6).d(C13-epsilon A14-C15) segment establishes that the dG5(anti).epsilon dA14(syn) alignment is stabilized by two hydrogen bonds from the NH1 and NH2-2 of dG5(anti) to N9 and N1 of epsilon dA14(syn), respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Deoxyguanosine-deoxyadenosine pairing in the d(C-G-A-G-A-A-T-T-C-G-C-G) duplex: conformation and dynamics at and adjacent to the dG X dA mismatch site

Nuclear magnetic resonance (NMR) has been used to monitor the conformation and dynamics of the d-(C1-G2-A3-G4-A5-A6-T6-T5-C4-G3-C2-G1) self-complementary dodecanucleotide (henceforth called 12-mer GA) that contains a dG X dA purine-purine mismatch at position 3 in the sequence. These results are compared with the corresponding d(C-G-C-G-A-A-T-T-C-G-C-G) dodecamer duplex (henceforth called 12-mer) containing standard Watson-Crick base pairs at position 3 [Patel, D.J., Kozlowski, S.A., Marky, L.A., Broka, C., Rice, J.A., Itakura, K., & Breslauer, K.J. (1982) Biochemistry 21, 428-436]. The dG X dA interaction at position 3 was monitored at the guanosine exchangeable H-1 and nonexchangeable H-8 protons and the nonexchangeable adenosine H-2 proton. We demonstrate base-pair formation between anti orientations of the guanosine and adenosine rings on the basis of nuclear Overhauser effects (NOE) observed between the H-2 proton of adenosine 3 and the imino protons of guanosine 3 (intra base pair) and guanosines 2 and 4 (inter base pair). The dG(anti) X dA(anti) pairing should result in hydrogen-bond formation between the guanosine imino H-1 and carbonyl O-6 groups and the adenosine N-1 and NH2-6 groups, respectively. The base pairing on either side of the dG X dA pair remains intact at low temperature, but these dG X dC pairs at positions 2 and 4 are kinetically destabilized in the 12-mer GA compared to the 12-mer duplex. We have estimated the hydrogen exchange kinetics at positions 4-6 from saturation-recovery measurements on the imino protons of the 12-mer GA duplex between 5 and 40 degrees C. The measured activation energies for imino proton exchange in the 12-mer GA are larger by a factor of approximately 2 compared to the corresponding values in the 12-mer duplex. This implies that hydrogen exchange in the 12-mer GA duplex results from a cooperative transition involving exchange of several base pairs as was previously reported for the 12-mer containing a G X T wobble pair at position 3 [Pardi, A., Morden, K.M., Patel, D.J., & Tinoco, I., Jr. (1982) Biochemistry 21, 6567-6574]. We have assigned the nonexchangeable base protons by intra and inter base pair NOE experiments and monitored these assigned markers through the 12-mer GA duplex to strand transition.(ABSTRACT TRUNCATED AT 400 WORDS)     

Solution conformation of the (+)-trans-anti-benzo[g]chrysene-dA adduct opposite dT in a DNA duplex.

The solution structure of the adduct derived from the covalent bonding of the fjord region (+)-(11S, 12R, 13R, 14S) stereoisomer of anti -11,12-dihydroxy-13,14-epoxy-11,12,13, 14-tetrahydrobenzo[g]chrysene, (+)- anti -B[g]CDE, to the exocyclic N(6)amino group of the adenine residue dA6, (designated (+)- trans-anti -(B[g]C)dA6), positioned opposite a thymine residue dT17 in the DNA sequence context d(C1-T2-C3-T4-C5-(B[g]C)A6-C7-T8-T9-C10-C11). d(G12-G13-A14-A15-G16-T17-G18-A19-G20++ +-A21-G22) (designated (B[g]C)dA. dT 11-mer duplex), has been studied using structural information derived from NMR data in combination with molecular dynamics (MD) calculations. The solution structure of the (+)- trans-anti -(B[g]C)dA.dT 11-mer duplex has been determined using an MD protocol where both interproton distance and dihedral angle restraints deduced from NOESY and COSY spectra are used during the refinement process, followed by additional relaxation matrix refinement to the observed NOESY intensities to account for spin diffusion effects. The results established that the covalently attached benzo[g]chrysene ring intercalates into the DNA helix directed towards the 5'-side of the modified strand and stacks predominantly with dT17 when intercalated between dC5.dG18 and (B[g]C)dA6.dT17 base-pairs. All base-pairs, including the modified (B[g]C)dA6.dT17 base-pair, are aligned through Watson-Crick pairing as in normal B -DNA. In addition, the potential strain associated with the highly sterically hindered fjord region of the aromatic portion of the benzo[g]chrysenyl ring is relieved through the adoption of a non-planar, propeller-like geometry within the chrysenyl ring system. This conformation shares common structural features with the related (+)- trans-anti -(B[c]Ph)dA adduct in the identical base sequence context, derived from the fjord region (+)-(1S,2R,3R,4S)-3, 4-dihydroxy-1,2-epoxy-1,2,3,4-tetrahydrobenzo[c]phenanthrene stereoisomer, in which intercalation is also observed towards the 5'-side of the modified dA6.dT17 base-pair.     

Conformation, dynamics, and structural transitions of the TATA box region of self-complementary d[(C-G)n-T-A-T-A-(C-G)n] duplexes in solution

Structural and kinetic features of the TATA box located in the center of the alternating self-complementary d(C-G-C-G-T-A-T-A-C-G-C-G) duplex (TATA 12-mer) and d(C-G-C-G-C-G-T-A-T-A-C-G-C-G-C-G) duplex (TATA 16-mer) have been probed by high-resolution proton and phosphorus NMR spectroscopy in aqueous solution. The imino exchangeable Watson-Crick protons and the nonexchangeable base protons in the TATA box of the TATA 12-mer and TATA 16-mer duplexes have been assigned from intra and inter base pair nuclear Overhauser effect (NOE) measurements. Imino proton line-width and hydrogen exchange saturation recovery measurements demonstrate that the dA X dT base pairs in the TATA box located in the center of the TATA 12-mer and TATA 16-mer duplexes are kinetically more labile than flanking dG X dC base pairs. The proton and phosphorus NMR parameters of the TATA 12-mer monitor a cooperative premelting transition in the TATA box prior to the onset of the melting transition to unstacked strands. Phosphorus NMR studies have been unable to detect any indication of a right-handed B DNA to a left-handed Z DNA transition for the TATA 12-mer duplex in saturated NaCl solution. By contrast, we do detect the onset of the B to Z transition for the TATA 16-mer in saturated NaCl solution. Proton and phosphorus NMR studies demonstrate formation of a loop conformation with chain reversal at the TATA segment for the TATA 12-mer and TATA 16-mer duplexes on lowering the DNA and counterion concentration. The imino protons (10-11 ppm) and phosphorus resonances (3.5-4.0 ppm; 4.5-5.0 ppm) of the loop segment fall in spectral windows well resolved from the corresponding markers in fully paired segments so tha it should be possible to identify loops in longer DNA helixes. The equilibrium between the loop and fully paired duplex conformations of the TATA 12-mer and TATA 16-mer is shifted toward the latter on addition of moderate salt.     

DNA fragment conformations. A 1H-NMR conformational analysis of the d(G-G)-chelated platinum-oligonucleotide d(A-T-G-G)cisPt

The conformation of d(A-T-G-G) and d(A-T-G-G)cisPt has been investigated by 1H-NMR at 500 MHz and 90 MHz under various experimental conditions of temperature and concentration. Analysis of the coupling constants between the deoxyribose protons shows that all the sugar rings of d(A-T-G-G) adopt the S(C2'-endo) conformation most of the time. By contrast, in the platinated tetramer, d(A-T-G-G)cisPt, the N(C3'-endo) conformation is highly predominant for the internal dG residue while the S(C2'-endo) conformation is largely favoured for the other residues as in the case of the unplatinated compound. The relaxation time and nuclear Overhauser effect measurements indicate that the orientation of the two guanines of d(A-T-G-G)cisPt is anti in agreement with the previous results obtained for the dimers: r(G-G)cisPt, d(G-G)cisPt. On lowering the temperature from 80 degrees C to 20 degrees C, several proton resonances of d(A-T-G-G)cisPt exhibit large chemical shift and linewidth variations. The most spectacular temperature effect was observed for the internal dG(H1') and dT(H4') protons. All the delta = f(t) curves display a sigmoid form with the same mid-point temperature of 44 +/- 2 degrees C. This mid-point temperature together with the observed chemical shift and linewidth variations were found to be independent of the d(A-T-G-G)cisPt concentration. These results suggest that d(A-T-G-G)cisPt can adopt two different conformations depending on the temperature. The enthalpy for the transition between the high and low temperature conformations is about 84 kJ/mol.     

Solution structures of aminofluorene [AF]-stacked conformers of the syn [AF]-C8-dG adduct positioned opposite dC or dA at a template-primer junction.

A solution structural study has been undertaken on the aminofluorene-C8-dG ([AF]dG) adduct located at a single-strand-double-strand d(A1-A2-C3-[AF]G4-C5-T6-A7-C8-C9-A10-T11-C12-C13). d(G14-G15-A16-T17-G18-G19-T20- A21-G22-N23) 13/10-mer junction (N = C or A) using proton-proton distance restraints derived from NMR data in combination with intensity-based relaxation matrix refinement computations. This single-strand-double-strand junction models one arm of a replication fork composed of a 13-mer template strand which contains the [AF]dG modification site and a 10-mer primer strand which has been elongated up to the modified guanine with either its complementary dC partner or a dA mismatch. The solution structures establish that the duplex segment retains a minimally perturbed B-DNA conformation with Watson-Crick hydrogen-bonding retained up to the dC5.dG22 base pair. The guanine ring of the [AF]dG4 adduct adopts a syn glycosidic torsion angle and is displaced into the major groove when positioned opposite dC or dA residues. This base displacement of the modified guanine is accompanied by stacking of one face of the aminofluorene ring of [AF]dG4 with the dC5.dG22 base pair, while the other face of the aminofluorene ring is stacked with the purine ring of the nonadjacent dA2 residue. By contrast, the dC and dA residues opposite the junctional [AF]dG4 adduct site adopt distinctly different alignments. The dC23 residue positioned opposite the adduct site is looped out into the minor groove by the aminofluorene ring. The syn displaced orientation of the modified dG with stacking of the aminofluorene and the looped out position of the partner dC could be envisioned to cause polymerase stalling associated with subsequent misalignment leading to frameshift mutations in appropriate sequences. The dA23 residue positioned opposite the adduct site is positioned in the major groove with its purine ring aligned face down over the van der Waals surface of the major groove and its amino group directed toward the T6.A21 base pair. The Hoogsteen edge of the modified guanine of [AF]dG4 and the Watson-Crick edge of dA23 positioned opposite it are approximately coplanar and directed toward each other but are separated by twice the hydrogen-bonding distance required for pairing. This structure of [AF]dG opposite dA at a model template-primer junctional site can be compared with a previous structure of [AF]dG opposite dA within a fully paired duplex [Norman, D., Abuaf, P., Hingerty, B. E., Live, D. , Grunberger, D., Broyde, S., and Patel, D. J. (1989) Biochemistry 28, 7462-7476]. The alignment of the Hoogsteen edge of [AF]dG (syn) positioned opposite the Watson-Crick edge of dA (anti) has been observed for both systems with the separation greater in the case of the junctional alignment in the model template-primer system. However, the aminofluorene ring is positioned in the minor groove in the fully paired duplex while it stacks over the junctional base pair in the template-primer system. This suggests that the syn [AF]dG opposite dA junctional alignment can be readily incorporated within a duplex by a translation of this entity toward the minor groove.     

NMR structural studies of the ionizing radiation adduct 7-hydro-8-oxodeoxyguanosine (8-oxo-7H-dG) opposite deoxyadenosine in a DNA duplex. 8-Oxo-7H-dG(syn).dA(anti) alignment at lesion site

Proton NMR studies are reported on the complementary d(C1-C2-A3-C4-T5-A6-oxo-G7-T8-C9-A10-C11-C12).d(G13-G14-T15- G16-A17-A18-T19- A20-G21-T22-G23-G24) dodecanucleotide duplex (designated 8-oxo-7H-dG.dA 12-mer), which contains a centrally located 7-hydro-8-oxodeoxyguanosine (8-oxo-7H-dG) residue, a group commonly found in DNA that has been exposed to ionizing radiation or oxidizing free radicals. From the NMR spectra it can be deduced that this moiety exists as two tautomers, or gives rise to two DNA conformations, that are in equilibrium and that exchange slowly. The present study focuses on the major component of the equilibrium that originates in the 6,8-dioxo tautomer of 8-oxo-7H-dG. We have assigned the exchangeable NH1, NH7, and NH2-2 base protons located on the Watson-Crick and Hoogsteen edges of 8-oxo-7H-dG7 in the 8-oxo-7H-dG.dA 12-mer duplex, using an analysis of one- and two-dimensional nuclear Overhauser enhancement (NOE) data in H2O solution. The observed NOEs derived from the NH7 proton of 8-oxo-7H-dG7 to the H2 and NH2-6 protons of dA18 establish an 8-oxo-7H-dG7(syn).dA 18(anti) alignment at the lesion site in the 8-oxo-7H-dG.dA 12-mer duplex in solution. This alignment, which places the 8-oxo group in the minor groove, was further characterized by an analysis of the NOESY spectrum of the 8-oxo-7H-dG.dA 12-mer duplex in D2O solution. We were able to detect a set of intra- and interstrand NOEs between protons (exchangeable and nonexchangeable) on adjacent residues in the d(A6-oxo-G7-T8).d(A17-A18-T19) trinucleotide segment centered about the lesion site that establishes stacking of the oxo-dG7(syn).dA(anti) pair between stable Watson-Crick dA6.dT19 and dT8.dA17 base pairs with minimal perturbation of the helix. Thus, both strands of the 8-oxo-7H-dG.dA 12-mer duplex adopt right-handed conformations at and adjacent to the lesion site, the unmodified bases adopt anti glycosidic torsion angles, and the bases are stacked into the helix. The energy-minimized conformation of the central d(A6-oxo-G7-T8).d(A17-A18-T19) segment requires that the 8-oxo-7H-dG7(syn).dA18(anti) alignment be stabilized by two hydrogen bonds from NH7 and O6 of 8-oxo-7H-dG7(syn) to N1 and NH2-6 of dA18(anti), respectively, at the lesion site.(ABSTRACT TRUNCATED AT 400 WORDS)     

Alternating d(G-C)3 and d(C-G)3 hexanucleotides containing 7-deaza-2''-deoxyguanosine or 8-aza-7-deaza-2''-deoxyguanosine in place of dG.

The synthesis of alternating hexamers (8-13) derived from d(C-G)3 or d(G-C)3 but containing c7z8Gd (2) or c7Gd (3) instead of dG is described employing phosphoramidite-chemistry. Apart from the isobutyryl group the dimethylaminomethylene residue was used for the nucleobase-protection of 3. The methyl- and the cyanoethyl-phosphoramidites of 3 (5a-c) were synthesized. They were employed together with those of c7G or c7z8Gd in automated oligonucleotide synthesis. Tm-values as well as thermodynamic data of the oligomers 9, 10, 12, and 13 indicated that duplexes were destabilized if c7Gd replaced dG, whereas c7z8Gd stabilized the duplex structure. In contrast to d(C-G)3 which underwent salt-dependent B-Z transition, CD-spectra of oligomers containing c7Gd or c7z8Gd in place of dG showed retained B-conformation.     

Solution structure of an O6-[4-oxo-4-(3-pyridyl)butyl]guanine adduct in an 11 mer DNA duplex: evidence for formation of a base triplex

The pyridyloxobutylating agents derived from metabolically activated tobacco-specific nitrosamines can covalently modify guanine bases in DNA at the O(6) position. The adduct formed, O(6)-[4-oxo-4-(3-pyridyl)butyl]guanine ([POB]dG), results in mutations that can lead to tumor formation, posing a significant cancer risk to humans exposed to tobacco smoke. A combined NMR-molecular mechanics computational approach was used to determine the solution structure of the [POB]dG adduct within an 11mer duplex sequence d(CCATAT-[POB]G-GCCC).d(GGGCCATATGG). In agreement with the NMR results, the POB ligand is located in the major groove, centered between the flanking 5'-side dT.dA and the 3'-side dG.dC base pairs and thus in the plane of the modified [POB]dG.dC base pair, which is displaced slightly into the minor groove. The modified base pair in the structure adopts wobble base pairing (hydrogen bonds between [POB]dG(N1) and dC(NH4) amino proton and between [POB]dG(NH2) amino proton and dC(N3)). A hydrogen bond appears to occur between the POB carbonyl oxygen and the partner dC's second amino proton. The modified guanine purine base, partner cytosine pyrimidine base, and POB pyridyl ring form a triplex via this unusual hydrogen-bonding pattern. The phosphodiester backbone twists at the lesion site, accounting for the unusual phosphorus chemical shift differences relative to those for the control DNA duplex. The helical distortions and wobble base pairing induced by the covalent binding of POB to the O(6)-position of dG help explain the significant decrease of 17.6 degrees C in melting temperature of the modified duplex relative to the unmodified control.     

Structural studies of the O6meG.C interaction in the d(C-G-C-G-A-A-T-T-C-O6meG-C-G) duplex

One- and two-dimensional nuclear magnetic resonance (NMR) experiments have been undertaken to investigate the conformation of the d(C1-G2-C3-G4-A5-A6-T7-T8-C9-O6meG10-C11-G12) self-complementary dodecanucleotide (henceforth called O6meG.C 12-mer), which contains C3.O6meG10 interactions in the interior of the helix. We observe intact base pairs at G2.C11 and G4.C9 on either side of the modification site at low temperature though these base pairs are kinetically destabilized in the O6meG.C 12-mer duplex compared to the G.C 12-mer duplex. One-dimensional nuclear Overhauser effects (NOEs) on the exchangeable imino protons demonstrate that the C3 and O6meG10 bases are stacked into the helix and act as spacers between the flanking G2.C11 and G4.C9 base pairs. The nonexchangeable base and H1', H2', H2', H3', and H4' protons have been completely assigned in the O6meG.C 12-mer duplex at 25 degrees C by two-dimensional correlated (COSY) and nuclear Overhauser effect (NOESY) experiments. The observed NOEs and their directionality demonstrate that the O6meG.C 12-mer is a right-handed helix in which the O6meG10 and C3 bases maintain their anti conformation about the glycosidic bond at the modification site. The NOEs between the H8 of O6meG10 and the sugar protons of O6meG10 and adjacent C9 exhibit an altered pattern indicative of a small conformational change from a regular duplex in the C9-O6meG10 step of the O6meG.C 12-mer duplex. We propose a pairing scheme for the C3.O6meG10 interaction at the modification site. Three phosphorus resonances are shifted to low field of the normal spectral dispersion in the O6meG.C 12-mer phosphorus spectrum at low temperature, indicative of an altered phosphodiester backbone at the modification site. These NMR results are compared with the corresponding parameters in the G.C 12-mer, which contains Watson-Crick base pairs at the same position in the helix.     

A parallel stranded linear DNA duplex incorporating dG.dC base pairs

DNA oligonucleotides with appropriately designed complementary sequences can form a duplex in which the two strands are paired in a parallel orientation and not in the conventional antiparallel double helix of B-DNA. All parallel stranded (ps) molecules reported to date have consisted exclusively of dA.dT base pairs. We have substituted four dA.dT base pairs of a 25-nt parallel stranded linear duplex (ps-D1.D2) with dG.dC base pairs. The two strands still adopt a duplex structure with the characteristic spectroscopic properties of the ps conformation but with a reduced thermodynamic stability. Thus, the melting temperature of the ps duplex with four dG.dC base pairs (ps-D5.D6) is 10-16 degrees C lower and the van't Hoff enthalpy difference delta HvH for the helix-coil transition is reduced by 20% (in NaCl) and 10% (in MgCl2) compared to that of ps-D1.D2. Based on energy minimizations of a ps-[d(T5GA5).d(A5CT5)] duplex using force field calculations we propose a model for the conformation of a trans dG.dC base pair in a ps helix.     

NMR studies for identification of dI:dG mismatch base-pairing structure in DNA.

One- and two-dimensional NMR experiments have been undertaken to investigate deoxyinosine:deoxyguanosine (dI:dG) base pairing in a self-complementary dodecadeoxyribonucleotide, d(C1-G2-C3-I4-A5-A6-T7-T8-G9-G10-G11-G12) (designated IG-12), duplex. The NMR data indicate formation of a dI(syn):dG(anti) base pair in a B-DNA helix. This unusual base pairing results in altered NOE patterns between the base protons (H8 and H2) of the I4 residue and the sugar protons of its own and the 5'-flanking C3 residues. The dI(syn):dG(anti) base pair is accommodated in the B-DNA duplex with only a subtle distortion of the local conformation. Identification of the dI:dG base pairing in this study confirms that a hypoxanthine base can form hydrogen-bonded base pairs with all of the four normal bases, C, A, T, and G, in DNA.     

Bulge-out structures in the single-stranded trimer AUA and in the duplex (CUGGUGCGG).(CCGCCCAG). A model-building and NMR study

Model-building studies were carried out on the trimer AUA. Bulge-out structures which allow incorporation into a continuous RNA helix were generated and energy-minimized. All geometrical features obtained by previous NMR studies on purine-pyrimidine-purine sequences are accounted for in these models. One of the models was used to fit into a double helical fragment. Only minor changes were necessary to construct a central bulge-out in an otherwise intact duplex. NMR and model-building studies were performed on the duplex (CUGGUGCGG).(CCGCCCAG) which contains an unpaired uridine residue. NOE data, chemical-shift profiles and imino-proton resonances provided evidence that the extra U is bulged out of the duplex. The relatively small dispersion in 31P chemical shifts (approximately equal to 0.7 ppm) indicate the absence of t/g or g/t combinations for the phosphodiester angles zeta/alpha. An energy-minimized model of the duplex, which fits the present collection of data, is presented.