Structural study of DNA duplex containing an N-(2-deoxy-beta-D-erythro-pentofuranosyl) formamide frameshift by NMR and restrained molecular dynamics.

The presence of an N-(2-deoxy-beta-D-erythro-pentofuranosyl) formamide (F) residue, a ring fragmentation product of thymine, in a frameshift context in the sequence 5'-d-(AGGACCACG)*d(CGTGGFTCCT) has been studied by 1H and 31P nuclear magnetic resonance (NMR) and molecular dynamics. Two-dimensional NMR studies show that the formamide residue, whether the cis or trans isomer, is rotated out of the helix and that the bases on either side of the formamide residue in the sequence, G14 and T16, are stacked over each other in a way similar to normal B-DNA. The cis and trans isomers were observed in the ratio 3:2 in solution. Information extracted from 31P NMR data reveal a modification of the phosphodiester backbone conformation at the extrahelical site, which is also observed during the molecular dynamics simulations.

Solution structure as a function of pH of two central mismatches, C . T and C . C, in the 29 to 39 K-ras gene sequence, by nuclear magnetic resonance and molecular dynamics.

The DNA duplexes 5' d(GCCACCAGCTC) x d(GAGCTXGTGGC), where the base X is either cytosine or thymine, have been studied by one and two-dimensional nuclear magnetic resonance, energy minimization and molecular dynamics. The sequence studied corresponds to the region 29 to 39 of the K-ras gene and is a hot spot for mutations. The results show that both duplexes adopt a globally B-DNA-type structure. For the C x C mismatch, we observe a structural change as a function of pH with an apparent pK of 6.95. The neutral species has only one hydrogen bond between the two bases but shows two families of wobble structures where one base or the other is displaced in the major groove. The protonated species has two hydrogen bonds and two structures but of unequal populations. In both systems, the sugar puckers remain predominantly C2'-endo and no significant changes in the backbone structure are observed. The neutral C . T mismatch is stabilized by two hydrogen bonds but, surprisingly, it can also be protonated, although the apparent pK is much lower, 5.65. In this case, protonation does not result in an additional hydrogen bond but must be due to better base-stacking interactions for C+ x T. The NMR data show that the environment of the T imino proton is very similar for C x T and C+ x T, although the hydrogen bond acceptor would be expected to be a nitrogen atom in the former case and an oxygen atom in the latter. We propose that for both structures there is an intervening water molecule which in addition reduces backbone strain. We have also measured the fluctuations during molecular dynamics runs in these mismatches. All are greater than for Watson-Crick base-pairs and the C x C mismatch shows very pronounced mobility.

Helical parameters, fluctuations, alternative hydrogen bonding, and bending in oligonucleotides containing a mistmatched base-pair by NOESY distance restrained and distance free molecular dynamics.

We have analysed and compared the molecular structures and dynamics of the DNA duplex, that corresponds to the sequence 29 to 39 of the K-ras gene, where the central base-pair is the normal C.G base or a mismatch base-pair C.A, C.A+, A.G and A+.G. Molecular dynamics runs of 100 picoseconds without or with distance restraints derived from NOE measurements are analysed and compared in all cases. (1) The EMBO convention of helical parameters for nucleic acids is extended to account for the construction and the description of any DNA mismatched base-pair. (2) Both types of MD runs reproduce very well all NMR data, except the H8 H2 inter-residue distances where agreement is not as good. (3) Average parameter values and fluctuations are in good agreement with results derived from persistence length and torsion modulus measurements. (4) Our molecular dynamics suggest the presence, in certain cases, of three-centred hydrogen bonds, which should be viewed as an equilibrium between hydrogen-bonding alternatives. In the case of the C.A mismatch, we observe the correlation between transient DNA bending and possible hydrogen bonding between a base and its 5' neighbour on the opposite strand in the sequence CCA. (5) These molecular dynamics analyses and observations provide a coherent view for the results obtained from recent DNA crystal structures and for results derived from other techniques such as gel electrophoresis at C.A steps.

Base-pair induced shifts in the tautomeric equilibrium of a modified DNA base.

We have examined the base-pairing properties of N4-methoxycytosine (mo4C), a mutagenic base analog, in DNA by nuclear magnetic resonance spectroscopy. Unlike standard bases, the tautomeric equilibrium of mo4C could be strongly influenced by base-pair formation. Paired with A, mo4C is found predominantly in the imino configuration in Watson-Crick geometry. However, when paired with G, two structurally distinct configurations are observed in equilibrium with one another. In one configuration, mo4C is in the amino form paired with G in Watson-Crick geometry. In the second species, mo4C is in the imino configuration paired with G in a wobble geometry. This is the first demonstration of basepair induced tautomeric shifts in DNA and supports the hypothesis that rare tautomeric forms may be involved in mutagenesis.

Solution structure of an oncogenic DNA duplex, the K-ras gene and the sequence containing a central C.A or A.G mismatch as a function of pH: nuclear magnetic resonance and molecular dynamics studies.

The DNA duplex 5' d(GCCACCAGCTC)-d(GAGCTGGTGGC) corresponds to the sequence 29 to 39 of the K-ras gene, which contains a hot spot for mutations. This has been studied by one and two-dimensional nuclear magnetic resonance, energy minimization and molecular dynamics. The results show that it adopts a globally B-DNA type structure. We have introduced, at the central base-pair, the mismatches C.A and A.G. The mismatch position is that of the first base of the Gly12 codon, the hot spot. For the C.A mismatch we observe a structural change as a function of pH with an apparent pKa of 7.2. At low pH, the mismatch pair adopts a structure close to a classic wobble conformation with the cytidine residue displaced into the major groove. It is stabilised by two hydrogen bonds in which the adenosine residue is protonated and the cytidine residue has a significant C3'-endo population. At high pH, the mispair structure is in equilibrium between wobble and reverse wobble conformations. Similar studies are reported on the A.G mismatch, which also undergoes a transition as a function of pH. 31P spectra have been recorded on all systems and as a function of pH. No evidence for BII phosphodiester backbone conformations was found. The NMR results are well corroborated by molecular dynamics calculations performed with or without distance constraints. The dynamics at the mismatch sites have been examined. Although the overall structures are close to B-DNA, helical parameters fluctuate differently at these sites. Different hydrogen bonding alternatives in dynamic equilibrium that can involve three-centred hydrogen bonds are observed.

The abasic site as a challenge to DNA polymerase. A nuclear magnetic resonance study of G, C and T opposite a model abasic site.

An abasic site in DNA creates a strong block to DNA polymerase and is a mutagenic base lesion. In this study, we present structural and dynamic properties of duplex oligodeoxynucleotides containing G, C and T opposite a model abasic site studied by one and two-dimensional nuclear magnetic resonance spectroscopy. We have demonstrated that A opposite the abasic site was positioned within the helix as if paired with T, and that the A residue melted co-operatively with the surrounding helix. We report here that G opposite the abasic site is also observed to be predominantly intrahelical in a normal anti conformation at low temperature. With increasing temperature, the mobility of the G residue increases rapidly and apparently is in a "melted state" well before denaturation of the helix. At low temperature, two species are found for T opposite the abasic site; one, intrahelical, one extrahelical. These species are in slow exchange with one another on a proton nuclear magnetic resonance time-scale. The two species then move into fast exchange with increasing temperature and the proportion of the extra-helical form increases. When C is positioned opposite the abasic site, both the C residue and the abasic sugar are extrahelical, the helix collapses, and the adjacent G.C base-pairs stack over one another. On the basis of these observations, we propose a model that explains why the abasic site acts to block DNA replication. Further, we suggest an explanation for the observed polymerase preference for base selection at abasic sites.

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.

A----Z transition in the synthetic hexanucleotide (dCdGfl)3.

500 MHz proton NMR and NOE measurements on (dCdGfl)3 show that at very low ionic strength the hexanucleotide adopts an A-DNA conformation, whereas at high salt concentrations a Z-form is found. At intermediate salt concentrations the two species are in slow exchange on the proton NMR time scale. This transition was also observed by characteristic changes in the CD spectra.

A proton 2D-NMR study of an oligodeoxyribonucleotide containing N6-methyladenine:d(GGm6ATATCC).

The presence of a m6A-T base pair does not give rise to a major change in helix conformation from that of a normal B DNA, but does slow down dramatically the rate of helix formation.

Structure of oligonucleotides with either a strand break or a bulged nucleotide.

We report NMR and molecular modelling studies on a DNA duplex structure which is composed of three oligonucleotides and mimics a strand break. Although it retains a B form conformation our model suggests that it is kinked at the strand break. In the same sequence with an extra bulged adenosine at the centre for the major species this residue is stacked in the helix and a kink is observed in the model.

Structure of an oligonucleotide containing a N-(2-deoxy-beta-D-erythro-pentofuranosyl)formamide residue facing a guanine.

Formamide residue is a major oxidative DNA damage product from ionizing radiation on thymine residues in DNA. We report NMR and molecular modeling studies on a DNA duplex structure which contains guanine opposite formamide residue. Formamide residue exists as either the cis and trans isomer. For the trans and the cis isomers, we find that guanine and formamide are stacked inside the helix and are hydrogen bonded. The oligonucleotide adopts globally a B form structure for the two isomers. Conformational changes are observed between the two isomers.

Methoxyamine attack on cytosine produces ambivalent base pairing properties of the modified base.

We report the solution structure of two heptanucleotides each containing a central N4-methoxycytosine, in one case with adenine on the opposite strand and in the other with guanine. For the N4-methoxycytosine-adenine pair only the imino form of the N4-methoxycytosine residue is observed and base pairing is in Watson-Crick geometry. However, rotation of the methoxy group about the N-OCH3 bond is not constrained to a particular orientation although it must be anti to the N3 of N4-methoxycytosine. The slow exchange on a proton NMR time scale between the single strand and double strand forms is attributed to the strong preference of the syn conformation of the OCH3 group in the single strand which inhibits base pair formation. For N4-methoxycytosine base paired with guanosine we observe the N4-methoxycytosine base in the amino form in Watson-Crick geometry and a slow exchange of this species with an imino form base paired in wobble geometry. The amino form is predominant at low temperature whereas the imino form predominates above 40 degrees C. Our results point to preferential replacement of dTTP by N4-methoxycytosine in primer elongation.

Z-DNA is formed by poly (dC-dG) and poly (dm5C-dG) at micro or nanomolar concentrations of some zinc(II) and copper(II) complexes.

We report studies on the interaction of some zinc(II) and copper(II) complexes of amines and amino acids with poly(dC-dG) and poly(dm5C-dG). Of the zinc complexes the species zinc-tris(2-aminoethyl) amine is found to be the most efficient for inducing Z-DNA giving a mid point at low ionic strength of 1.4 microM (poly(dC-dG] and 44nM (poly(dm5C-dG). While an antagonistic effect on raising the ionic strength is observed, the transition occurs at only 2 microM for poly(dm5C-dG) at 150mM NaCl. The most efficient copper(II) complex is that of diethylene triamine, though copper(II) complexes are generally less efficient than zinc(II) complexes. We also report kinetic and thermodynamic studies upon the B-Z transition induced by these complexes. A model is proposed for the interaction of one of the zinc complexes which involves not only direct zinc-DNA binding but also the formation of hydrogen bonds between the metal bond amine groups and the residues adjacent to the coordination site.

Structures of oligonucleotides containing 5-(methoxymethyl)-2'-deoxyuridine determined by NMR spectroscopy.

Base pairing of 5-(methoxymethyl)-2'-deoxyuridine (MMdU) opposite either adenine or guanine in a seven base pair oligonucleotide duplex has been studied by NMR spectroscopy. When paired with A, we observe that the MMdU.A base pair adopts Watson-Crick geometry. The methoxymethyl substituent is not held in a fixed conformation and may rotate around the C5-CH2 and CH2-O bonds. Examination of the potential energy as a function of rotation around these bonds indicates the presence of four low energy conformations. No hydrogen bonding is indicated for the methoxymethyl substituent, and the four potential minima result from reduced steric clash. For the MMdU.G base pair, the two bases adopt a wobble geometry which does not change with increasing solvent pH. Similarly, we find four low energy conformations for the methoxymethyl substituent in the major groove of the DNA helix.

Molecular mechanics and dynamics of an abasic frameshift in DNA and comparison to NMR data.

In a previous publication (Ph. Cuniasse, L.C. Sowers, R. Eritja, B. Kaplan, M.F. Goodman, J.A.H. Cognet, M. Le Bret, W. Guschlbauer and G.V. Fazakerley, Biochemistry 28, 2018 (1989), we determined by two dimensional NMR studies and molecular mechanics calculations the three-dimensional structure of a non-selfcomplementary oligonucleotide: [sequence; see text] where dr, at the center of the first strand, is a model abasic site. In order to explain all the results arising from NMR measurements, we found that an equilibrium between two conformations was necessary. These conformations differ mainly by the sugar pucker of G5 which is C2' endo or C3' endo. The latter is stabilized by addition of counterions between phosphate residues P3 and P4. In this paper, we have constructed systematically, all possible structures as a function of torsion angles delta of dr4 and of G5 by molecular mechanics in the presence or absence of counterions. Since these conformations were not forced with NMR distance measurements, this method allows detailed comparisons between all possible conformations and NMR data. Maps of contour lines of the potential energy, of fits to NMR distance measurements, and of helical twist as a function of torsion angles delta of dr4 and of G5 unravel the difficulties associated with the study of the G5 sugar pucker conformation equilibrium. Sugar puckers and proton distances are very sensitive criteria to monitor molecular dynamics. Relying on these experimental criteria, we have tested many molecular dynamics preparation phases and we propose a new warm-up and equilibration procedure for molecular dynamics. Thus we show with a 290 ps molecular dynamic run that G5 is in conformational equilibrium and that all NMR data are well reproduced.

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

We have synthesized and studied by proton NMR a duplex heptaoligonucleotide containing a 5-bromouracil (brU)-adenine base pair. This represents the first structural characterization of a B-form DNA containing brU. The brU.A base pair is Watson-Crick rather than Hoogsteen as seen for the monomers in the crystalline state. From analysis of the NOESY sepctra at very short mixing times evidence is presented that substitution of brU for T induces significant conformational changes from that of a normal B DNA. The helix twist between brU4.A11 and G3.C12 is ca. 15 degrees and for both brU4 and G3 the glycosyl torsion angles are significantly changed. The imino proton of the bru.A base pair shows a pH insensitive line with which shows that the pK of brU in this base pair is very much higher than that of the monomer.

Metal-ion interaction with penicillins: kinetics of complexation of nickel(II).

The kinetics of complexation of nickel(II) with some penicillins and related compounds show that the zwitterionic form of the ligand has very low reactivity compared to the anionic form. The resolved rate constants are interpreted in terms of binding to the ring nitrogen and carboxyl group and not to the side chain.

Zinc Z-DNA.

Circular dichroism spectra of poly(dG-dC) in the presence of some zinc complexes exhibit the characteristic inversion associated with the formation of a left handed helix. The transition of B to Z DNA is cooperative and slow. The concentration of zinc complex at the mid point of the transition is strongly dependent upon the nature of the ligand bound to zinc. The most efficient species is one with a tetradentate amine for which the mid point is observed at a zinc:nucleotide ratio of 1:24. 31P spectra of one of these complexes confirm the presence of a left handed helix.

Determination of the interaction of ADP and dADP with copper(II), manganese(II) and lanthanide(III) ions by nuclear-magnetic-resonance spectroscopy.

The aqueous solution conformation of the 1:1 complexes of ADP and dADP bound to a lanthanide ion have been determined by examination of the dipolar shifts and induced relaxation at pH 6.4. Apparent inconsistencies in the observed data are interpreted in terms of a gradually changing lanthanide-oxygen bond length from Pr3+ to Yb3+. The conformations of ADP and dADP are very similar showing an extended diphosphate, a 2E ribose conformation and with the adenine base displaying a small syn contribution. Relaxation data obtained from Mn(II) titrations are readily interpreted in terms of bidentate coordination to alpha and beta phosphates with the nucleotides retaining the same overall conformation found from the lanthanide study. No evidence to support an intramolecular water-bridged backbound structure is observed. The interaction with Cu(II) is more complex, coordination is observed not only at the diphosphate but also at two sites on the base, N-1, and the chelate formed between N-7 and the amino group. The relative importance of these sites is different for ADP and dADP and is also pH-dependent for ADP.