Anomalous structure and properties of poly (dA).poly(dT). Computer simulation of the polynucleotide structure with the spine of hydration in the minor groove.

The results of the search for low-energy conformations of poly(dA).poly(dT) and of the poly(dA).poly(dT) "complex" with the spine of hydration similar to that found by Dickerson and co-workers (Kopka, M.L., Fratini, A.V., Drew, H.R. and Dickerson, R.E. (1983) J. Mol. Biol. 163, 129-146) in the minor groove of the CGCGAATTCGCG crystals are described. It is shown that the existence of such a spine in the minor groove of poly(dA).poly(dT) is energetically favourable. Moreover, the spine of hydration makes the polynucleotide conformation similar to the poly(dA).poly(dT) structure in fibers and to the conformation of the central part of CGCGAATTCGCG in crystals; it also acquires features characteristic of the structure of poly(dA).poly(dT) and DNA oligo(dA)-tracts in solution. It is shown that the existence of the TpA step in conformations characteristic of the poly(dA).poly(dT) complex with the spine of hydration is energetically unfavourable (in contrast to the ApT step) and therefore this step should result in destabilization of the spine of hydration in the DNA minor groove. Thus, it appears that the spine of hydration as described by Dickerson and co-workers is unlikely to exist in the poly d(A-T).poly d(A-T) structure. The data obtained permit us to interpret a large body of experimental facts concerning the unusual structure and properties of poly(dA).poly(dT) and oligo(dA)-tracts in DNA both in fibers and in solution. The results provide evidence of the existence of the minor groove spine of hydration both in fibers and in solution on A/T tracts of DNA which do not contain the TpA step. The spine plays an active role in the formation of the anomalous conformation of these tracts.     

Synergistic effects in the melting of DNA hydration shell: melting of the minor groove hydration spine in poly(dA).poly(dT) and its effect on base pair stability.

We propose that water of hydration in contact with the double helix can exist in several states. One state, found in the narrow groove of poly(dA).poly(dT), should be considered as frozen to the helix, i.e., an integral part of the double helix. We find that this enhanced helix greatly effects the stability of that helix against base separation melting. Most water surrounding the helix is, however, melted or disassociated with respect to being an integral part of helix and plays a much less significant role in stabilizing the helix dynamically, although these water molecules play an important role in stabilizing the helix conformation statically. We study the temperature dependence of the melting of the hydration spine and find that narrow groove nonbonded interactions are necessary to stabilize the spine above room temperature and to show the broad transition observed experimentally. This calculation requires that synergistic effects of nonbonded interactions between DNA and its hydration shell affect the state of water-base atom hydrogen bonds. The attraction of waters into narrow groove tends to retain waters in the groove and compress or strain these hydrogen bonds.     

Dependence of the hydration shell structure in the minor groove of the DNA double helix on the groove width as revealed by Monte Carlo simulation.

The hydration shell of several conformations of the polynucleotides poly(dA).poly(dT), poly(dA).poly(dU), and poly(dA-dI).poly(dT-dC) has been simulated using the Monte Carlo method (Metropolis sampling). Calculations have shown that the structure of the hydration shell of the minor groove greatly depends on its width. In conformations with a narrowed minor groove, the first layer of the hydration shell of this groove has only one molecule per nucleotide pair that forms H bonds with purine N3 of one pair and pyrimidine O2 of the next pair. The second layer of the hydration shell of such conformations contains molecules that form H bonds between two adjacent molecules of the first layer. The probability of formation of hydration spine is about 20% while the bridges of the first layer are formed with a probability of about 70%. In the first layer of the minor groove of the B-DNA conformation with wide minor groove there are approximately two water molecules per base pair that form H bonds with purine N3 or pyrimidine O2 and with the sugar ring oxygen of the adjacent nucleotide. The probability of simultaneous H bonding of a water molecule with N3 (or O2) and O of sugar ring is about 30%. The results of simulation suggest that hydration spine proposed for the narrowed minor groove of oligonucleotide crystals [H. R. Drew, and R. E. Dickerson (1981) Journal of Molecular Biology, Vol. 151, pp. 535-556] can be formed in fibers of poly(dA).poly(dT), poly(dA).poly(dU), and poly(dA-dI).poly(dT-dC) as well as in DNA fragments of these sequences in solution.(ABSTRACT TRUNCATED AT 250 WORDS)     

The propeller DNA conformation of poly(dA).poly(dT).

Physical properties of the DNA duplex, poly(dA).poly(dT) differ considerably from the alternating copolymer poly(dAT). A number of molecular models have been used to describe these structures obtained from fiber X-ray diffraction data. The recent solutions of single crystal DNA dodecamer structures with segments of oligo-A.oligo-T have revealed the presence of a high propeller twist in the AT regions which is stabilized by the formation of bifurcated (three-center) hydrogen bonds on the floor of the major groove, involving the N6 amino group of adenine hydrogen bonding to two O4 atoms of adjacent thymine residues on the opposite strand. Here we show that it is possible to incorporate the features of the single crystal analysis, specifically high propeller twist, bifurcated hydrogen bonds, and a narrow minor groove, as well as the close interstrand NMR signal between adenine HC2 and ribose HC1' of the opposite strand, into a model that is fully compatible with the diffraction data obtained from poly(dA).poly(dT).     

Nuclear Overhauser data and stereochemical considerations suggest that netropsin binds symmetrically within the minor groove of poly(dA).poly(dT), forming hydrogen bonds with both strands of the double helix

Sarma et al. (J. Biomol. Str. and Dynam. 2, 1085 (1985) have proposed, on the basis of nuclear magnetic resonance experiments on the complex of netropsin with poly(dA).poly(dT), that the drug molecule lies asymmetrically along the dA side of the minor groove and makes hydrogen bonds only with the dA strand. If the crystal structure analyses of B-DNA (Fratini et al., J. Biol. Chem. 257, 14686 (1982] and of its complex with netropsin (Kopka et al., J. Mol. Biol. 183, 553 (1985] are any guide, this off-center, wide-groove model is stereochemically unlikely. More to the point, the off-center model is unnecessary to explain the observed nmr data. All of the nuclear Overhauser and other observations are fully explained by the structure seen in the x-ray crystal analysis, in which netropsin sits squarely centered within the minor groove, making bifurcated hydrogen bonds with both strands.     

Different Effects of Nonintercalative Antitumor Drugs on DNA Triple Helix Stability: SN-18071 Promotes Triple Helix Formation

Abstract

The interaction of the nonintercalating bisquaternary ammonium heterocyclic drugs SN- 18071 and SN-6999 with a DNA triple helix has been studied using thermal denaturation and CD spectroscopy. Our data show, that both minor groove binders can bind to the triple helix of poly(dA)-2poly(dT) under comparable ionic conditions, but they influence the stability of the triplex relative to the duplex structure of poly(dA)-poly(dT) in a different manner. SN- 18071, a ligand devoid of forming hydrogen bonds, can promote triplex formation and thermally stabilizes it up to 500 mM Na+ concentration. SN-6999 destabilizes the triplex to duplex equibilirium whereas it stabilizes the duplex. The binding constant of SN-18071 is found to be greater than that to the duplex. The stabilizing effect of SN-18071 is explained by electrostatic inetractions of three ligand molecules with the three grooves of the triple stranded structure. From the experiments it is concluded that SN-6999 binds to the triplex minor groove thereby destabilizing the triplex similar as previously reported for netropsin.     

Cytosine methylation can induce local distortions in the structure of duplex DNA.

Methyl groups at the C5 position of pyrimidines located within oligopurine-oligopyrimidine tracts in DNA have been shown previously to modulate curvature generated by those tracts. However, it was not known whether the influence of such methyl groups is consequent to the altered helical structure within the tracts themselves. In the current study, it is demonstrated that methylation of cytosines up to three base pairs away from a (dA)5.(dT)5 tract (A-tract) can still result in alterations of the net curvature of the A-tract-containing DNA, as measured by alterations in electrophoretic mobility. This latter effect depends strongly on both the sequence of the non-A-tract DNA and the positions of the methylated C residues. The current results lend further support to the notion that the biological consequences of cytosine methylation may be effected through local alterations in DNA structure as well as through direct protein-DNA interactions.     

Flexibility of DNA in 2:1 drug-DNA complexes--simultaneous binding of two DAPI molecules to DNA.

Simultaneous binding of two DAPI molecules in the minor groove of (dA)15.(dT)15 B-DNA helix has been simulated by molecular mechanics calculations. The energy minimised structure shows some novel features in relation to binding of DAPI molecules as well as the flexibility of the grooves of DNA helices. The minor groove of the helix expands locally considerably (to 15 angstroms) to accommodate the two DAPI molecules and is achieved by positive propeller twisting of base pairs at the binding site concomitant with small variations in the local nucleotide stereochemistry. The expansion also brings forth simultaneously a contraction in the width of the major groove spread over to a few phosphates. These findings demonstrate another facet of the flexible stereochemistry of DNA helices in which the local features are significantly altered without being propagated beyond a few base pairs, and with the rest of the regions retaining the normal structure. Both the DAPI molecules are engaged in specific hydrogen bonds with the bases and non specific interactions with phosphates. Stacking interactions of DAPI molecules between themselves as well as with sugar-phosphate backbone contribute to the stability of the complex. The studies provide a stereochemical support to the experimental findings that under high drug-DNA ratio DAPI could bind in the 2:1 ratio.     

UV resonance Raman and circular dichroism studies of a DNA duplex containing an A(3)T(3) tract: evidence for a premelting transition and three-centered H-bonds.

The presence of A(n) and A(n)T(n) tracts in double-helical sequences perturbs the structural properties of DNA molecules, resulting in the formation of an alternate conformation to standard B-DNA known as B'-DNA. Evidence for a transition occurring prior to duplex melting in molecules containing A(n) tracts was previously detected by circular dichroism (CD) and calorimetric studies. This premelting transition was attributed to a conformational change from B'- to B-DNA. Structural features of A(n) and A(n)T(n) tracts revealed by X-ray crystallography include a large degree of propeller twisting of adenine bases, narrowed minor grooves, and the formation of three-centered H-bonds between dA and dT bases. We report UV resonance Raman (UVRR) and CD spectroscopic studies of two related DNA dodecamer duplexes, d(CGCAAATTTGCG)(2) (A(3)T(3)) and d(CGCATATATGCG)(2) [(AT)(3)]. These studies address the presence of three-centered H-bonds in the B' conformation and gauge the impact of these putative H-bonds on the structural and thermodynamic properties of the A(3)T(3) duplex. UVRR and CD spectra reveal that the premelting transition is only observed for the A(3)T(3) duplex, is primarily localized to the dA and dT bases, and is associated with base stacking interactions. Spectroscopic changes associated with the premelting transition are not readily detectable for the sugar-phosphate backbone or the cytosine and guanosine bases. The temperature-dependent concerted frequency shifts of dA exocyclic NH(2) and dT C4=O vibrational modes suggest that the A(3)T(3) duplex forms three-centered hydrogen bonds at low temperatures, while the (AT)(3) duplex does not. The enthalpy of this H-bond, estimated from the thermally induced frequency shift of the dT C4=O vibrational mode, is approximately 1.9 kJ/mol or 0.46 kcal/mol.     

Nuclear Overhauser Data and Stereochemical Considerations Suggest that Netropsin Binds Symmetrically Within the Minor Groove of Poly(dA)·Poly(dT), Forming Hydrogen Bonds with Both Strands of the Double Helix

Abstract

Sarma et al. (J. Biomol. Str. and Dynam. 2, 1085 (1985) have proposed, on the basis of nuclear magnetic resonance experiments on the complex of netropsin with poly(dA)·poly(dT), that the drug molecule lies asymmetrically along the dA side of the minor groove and makes hydrogen bonds only with the dA strand. If the crystal structure analyses of B-DNA (Fratini et al., J. Biol. Chem. 257, 14686 (1982)) and of its complex with netropsin (Kopka et al., J. Mol. Biol. 183, 553 (1985)) are any guide, this off-center, wide-groove model is stereochemically unlikely. More to the point, the off-center model is unnecessary to explain the observed nmr data. All of the nuclear Overhauser and other observations are fully explained by the structure seen in the x-ray crystal analysis, in which netropsin sits squarely centered within the minor groove, making bifurcated hydrogen bonds with both strands.     

A demonstration of the intrinsic importance of stabilizing hydrophobic binding and non-covalent van der Waals contacts dominant in the non-covalent CC-1065/B-DNA binding

The comparative DNA binding properties and cytotoxic activity of CDPIn methyl esters (n = 1-5) vs. PDE-In methyl esters (n = 1-3) are detailed in studies which provide experimental evidence for the intrinsic importance of stabilizing hydrophobic binding and non-covalent van der Waals contacts dominant in the CC-1065/B-DNA minor groove binding. High affinity minor groove binding to DNA was established through: (1) the observation of CDPI3 binding (UV) but not unwinding of supercoiled DNA (phi 174 RFI DNA) thus excluding intercalative binding; (2) the observation of CDPI3 binding to T4 phage DNA (UV, delta Tm) in which the major groove is occluded by glycosylation thus excluding major groove binding; (3) the observation of salt (Na+) concentration independent high affinity CDPI3 binding to poly(dA . poly(dT) thus excluding simple electrostatic binding to the DNA phosphate backbone; and further inferred through (4) the observation of an intense induced dichroism (ICD, poly(dA) . poly(dT) and poly(dG) . poly(dC) [phi]23(358) = 24,000 and 23,500). This high affinity minor groove binding is sufficient to produce a potent cytotoxic effect.(ABSTRACT TRUNCATED AT 400 WORDS)     

Quantitative structure of a complex between a minor-groove-specific drug and a bent DNA decamer duplex: use of 2D NMR data and NOESY constrained energy minimization

Two-dimensional nuclear magnetic resonance (2D NMR) studies on d(GA4T4C)2 and d(GT4A4C)2 [Sarma, M.H., et al. (1988) Biochemistry 27, 3423-3432; Gupta G., et al. (1988) Biochemistry 27, 7909-7919] showed that A.T pairs are propeller twisted. As a result, A/T tracts form a straight rigid structural block with an array of bifurcated inter base pair H bonds in the major groove. It was demonstrated (previous paper) that replacement of methyl group by hydrogen (changing from T to U) in the major groove does not disrupt the array of bifurcated H bonds in the major groove. In this article, we summarize results of 2D NMR and molecular mechanic studies on the effect of a minor-groove-binding A.T-specific drug on the structure d(GA4T4C)2. A distamycin analogue (Dst2) was used for this study. It is shown that Dst2 binds to the minor groove of d(GA4T4C)2 mainly driven by van der Waals interaction between A.T pairs and the drug; as a consequence, an array of bifurcated H bonds can be formed in the minor groove between amide/amino protons of Dst2 and A.T pairs of DNA. NOESY data suggest that Dst2 predominantly binds at the central 5 A.T pairs. NOESY data also reveal that, upon drug binding, d(GA4T4C)2 does not undergo any significant change in conformation from the free state; i.e., propeller-twisted A.T pairs are still present in DNA and hence the array of bifurcated H bonds must be preserved in the major groove. NOESY data for the A5-T6 sequence also indicate that there is little change in junction stereochemistry upon drug binding.     

Temperature-dependent ultraviolet resonance Raman spectroscopy of the premelting state of dA.dT DNA.

Poly(dA).poly(dT) and DNA duplex with four or more adenine bases in a row exhibits a broad, solid-state structural premelting transition at about 35 degrees C. The low-temperature structure is correlated with the phenomena of "bent DNA." We have conducted temperature-dependent ultraviolet resonance Raman measurements of the structural transition using poly(dA).poly(dT) at physiological salt conditions, and are able to identify, between the high and low temperature limits, changes in the vibrational frequencies associated with the C4 carbonyl stretching mode in the thymine ring and the N6 scissors mode of the amine in the adenine ring of poly(dA).poly(dT). This work supports the model that the oligo-dA tracts' solid-state structural premelting transition is due to a set of cross-stand bifurcated hydrogen bonds between consecutive dA. dT pairs.     

Differences in melting behavior between homopolymers and copolymers of DNA: Role of nonbonded forces for GC and the role of the hydration spine and premelting transition for AT

Melting measurements of the mono-base-pair DNA polymers showed that the melting temperature T_m of the B-DNA homopolymer poly (dA ) · poly (dT) is higher than that of the copolymer poly [d(A-T)]. On the other hand, the T_mof the B-DNA homopolymer poly (dG) · poly (dC) is lower than that of the copolymer poly [d (G-C)]. From a structural point of view, the cross-strand base-stacking interaction in a DNA homopolymer is weaker than that in a DNA copolymer with the same base pair. One would then expect that all the DNA homopolymers are less stable than the copolymer with the same base pair. We find that the inversion of the melting order seen in the AT mono-base-pair DNA polymers is caused by the enhanced thermal stability of poly (dA) · poly (dT) from a well-defined spine of hydration attached to its minor groove. In this paper we employ the modified self-consistent phonon theory to calculate base-pair opening probabilities of four B-DNA polymers: poly(dA)-poly(dT), poly(dG) · poly(dC), poly[d(A-T)], and poly[d(G-C)] at temperatures from room temperature through the melting regions. Our calculations show that the spine of hydration can give the inverted melting order of the AT polymers as compared to the GC polymers in fair agreement with experimental measurements. Our calculated hydration spine disruption behavior in poly(dA) · poly(dT) at premelting temperatures is also in agreement with experimentally observed premelting transitions in poly (dA) · poly (dT). The work is in a sense a test of the validity of our models of nonbonded interactions and spine of hydration interactions. We find we have to develop the concept of a strained bond to fit observations in poly (dA) · poly(dT). The strained-bond concept also explains the otherwise anomalous stability of the hydration chain. © 1993 John Wiley & Sons, Inc.     

Monte-Carlo Simulation of DNA Duplex Hydration. B and B′ Conformations of Poly(dA) · Poly(dT) Have Different Hydration Shells

Abstract

Monte-Carlo simulation of poly(dA) · poly(dT) hydration by 30 water molecules per nucleotide pair has been performed. Two B-family conformations, both with a 36° helical twist but with different minor groove widths, were considered. One conformation is Arnott's standard B form, the other one is specific for poly(dA) · poly(dT) B′ form with a narrowed minor groove. The mean energies and the mean numbers of water-water and water-DNA hydrogen bonds are close for the two conformations. Nevertheless, the hydration shell of the B' form differs drastically from that of the standard B form. The water arrangement in the minor groove of the B′ form resembles the spine of hydration in the central part of Dickerson's dodecamer d(CGCGAATTCGCG). No such spine is formed in the hydration shell of the usual B form with a wider minor groove. In this conformation water bridges between adenine N3 or thymine O2 and oxygen of the sugar ring of the neighbouring nucleotide along the chain can be formed (“strings” in Dickerson's decamer d(CCAAGATTGG)).     

Flexibility Of DNA In 2:1 Drug-DNA Complexes - Simultaneous Binding Of Two DAPI Molecules To DNA

Abstract

Simultaneous binding of two DAPI molecules in the minor groove of (dA)₁₅.(dT)₁₅ B-DNA helix has been simulated by molecular mechanics calculations. The energy minimised structure shows some novel features in relation to binding of DAPI molecules as well as the flexibility of the grooves of DNA helices. The minor groove of the helix expands locally considerably (to 15 Å) to accommodate the two DAPI molecules and is achieved by positive propeller twisting of base pairs at the binding site concomitant with small variations in the local nucleotide stereochemistry. The expansion also brings forth simultaneously a contraction in the width of the major groove spread over to a few phosphates. These findings demonstrate another facet of the flexible stereochemistry of DNA helices in which the local features are significantly altered without being propagated beyond a few base pairs, and with the rest of the regions retaining the normal structure. Both the DAPI molecules are engaged in specific hydrogen bonds with the bases and non specific interactions with phosphates. Stacking interactions of DAPI molecules between themselves as well as with sugar-phosphate backbone contribute to the stability of the complex. The studies provide a stereochemical support to the experimental findings that under high drug-DNA ratio DAPI could bind in the 2:1 ratio.     

Interaction of the nonintercalative antitumour drugs SN-6999 and SN-18071 with DNA: influence of ligand structure on the binding specificity

Binding to DNA's of the non-intercalative ligands SN-6999 and SN-18071 has been studied by means of circular dichroism, UV absorption, thermal melting and for SN-6999 by viscosity measurements. Both antitumour drugs show a preference for dA.dT rich DNA's, but the base pair selectivity of SN-18071 is lower as indicated by some affinity to dG.dC containing duplex DNA. The dA.dT base pair specificity of SN-6999 is comparable to that of netropsin. It forms very stable complexes with dA.dT containing duplex DNA and competes with netropsin binding on DNA. The ligands SN-18071 and pentamidine are totally released from their complexes with poly(dA-dT).poly(dA-dT) by competitive netropsin binding. The results demonstrate that hydrogen bonding capacity of the ligand in addition to other factors strongly contribute to the base sequence specificity in the recognition process of the ligand with DNA. A binding model of SN-6999 with five dA.dT pairs in the minor groove of B-DNA is suggested.     

Probing minor groove hydrogen bonding interactions between RB69 DNA polymerase and DNA

Minor groove hydrogen bonding (HB) interactions between DNA polymerases (pols) and N3 of purines or O2 of pyrimidines have been proposed to be essential for DNA synthesis from results obtained using various nucleoside analogues lacking the N3 or O2 contacts that interfered with primer extension. Because there has been no direct structural evidence to support this proposal, we decided to evaluate the contribution of minor groove HB interactions with family B pols. We have used RB69 DNA pol and 3-deaza-2'-deoxyadenosine (3DA), an analogue of 2-deoxyadenosine, which has the same HB pattern opposite T but with N3 replaced with a carbon atom. We then determined pre-steady-state kinetic parameters for the insertion of dAMP opposite dT using primer/templates (P/T)-containing 3DA. We also determined three structures of ternary complexes with 3DA at various positions in the duplex DNA substrate. We found that the incorporation efficiency of dAMP opposite dT decreased 10(2)-10(3)-fold even when only one minor groove HB interaction was missing. Our structures show that the HB pattern and base pair geometry of 3DA/dT is exactly the same as those of dA/dT, which makes 3DA an optimal analogue for probing minor groove HB interactions between a DNA polymerase and a nucleobase. In addition, our structures provide a rationale for the observed 10(2)-10(3)-fold decrease in the rate of nucleotide incorporation. The minor groove HB interactions between position n - 2 of the primer strand and RB69pol fix the rotomer conformations of the K706 and D621 side chains, as well as the position of metal ion A and its coordinating ligands, so that they are in the optinal orientation for DNA synthesis.     

New insights into the structure of An tracts and B'-B' bends in DNA

Energy calculations suggest that the currently available NOE distance constraints for An tracts in DNA are incapable of distinguishing between structures with a narrowed minor groove arising from a large propeller twist with a small inclination or from a small propeller twist with a large negative inclination. Furthermore, analysis of published data, together with energy estimations, strongly argue against bifurcated hydrogen bonding between A and T residues being the cause of the anomalous structural properties of An tracts. A conformational analysis of the B'-B' junction has been performed in which a single variable base pair has been inserted between two regions of B' structure. We have calculated low-energy structures for AnGAn,AnCAn,AnTAn,AnCTn, and TnCAn duplexes, where the An and Tn tracts were fixed in the anomalous B' conformation. Upon optimization, all these structures were found to contain a pronounced roll-like bending into the major groove at the site of the insertion. The important factors in the formation of these B'-B' bends are the destruction of the B' conformation and the concomitant widening of the minor groove at the junction region in order to reduce minor groove interstrand base clashes and improve interstrand stacking energy. If the B' conformation has strong negative inclination, the improved intrastrand stacking energy also contributes to the bending. In calculations of duplexes with An and Tn tracts in the B conformation instead of B', the bending disappears.     

Synthesis of a dA-dT base pair analogue and its effects on DNA-ligand binding

Two nucleoside derivatives containing the base analogues 3-deazaadenine and 3-methyl-2-pyridone have been prepared as analogues of dA and dT, respectively. After conversion into the appropriately protected phosphoramidites, DNA sequences were prepared with site-specifically placed analogues. When present in a duplex DNA sequence, the analogues result in the deletion of one or both of the hydrogen bonding functional groups (the N3-nitrogen of dA and the O2-carbonyl of dT) present in the minor groove. Binding by two ligands, 4',6-diamidine-2-phenyl indole (DAPI) and Hoechst 33258 in the minor groove has been probed using a variety of DNA sequences. These sequences contain a d(GAATTC)2 core with analogue nucleosides substituted for one or more of the dA and dT residues. DAPI bound strongly to any sequence that contained both O2-carbonyls of the central two dT residues. The presence of a dc3A residue did in some cases enhance binding. With one of the central O2-carbonyls deleted, the binding was noticeably reduced, and with both absent, no significant binding could be detected. Similar although less dramatic results were observed with Hoechst 33258 binding to analogue sequences.