A proposal for a specific double-helical structure in which the polynucleotide strands intercalate instead of forming base-pairs

Double helices, since the discovery of the DNA structure by Watson and Crick, represent the single most important secondary structural form of nucleic acids. The secondary structures of a variety of polynucleotide helices have now been well characterised with hydrogen-bonded base-pairs as building blocks. We wish to propose here the possibility, in a specific case, of a double stranded helical structure without any base-pair, but having a repeat unit of two nucleotides with their bases stacked through intercalation. The proposal comes from the initial models we have built for poly(dC) using the stacking patterns found in the crystal structures of 5'-dCMPNa2 which crystallises in two forms depending on the degree of hydration. These structures have pairs of nucleotides with the cytosine rings partially overlapping and separated by 3.3A. Using these as repeat units one could generate a model for poly(dC) with parallel strands, having a turn angle of 30 degrees and a base separation of 6.6A along each strand. Both right and left handed models with these parameters can be built in a smooth fashion without any obviously unreasonable stereochemical contacts. The helix diameter is about 13.5A, much smaller than that of normal helices with base-pair repeats. The changes in the sugar-phosphate backbone conformation in the present models compared to normal duplexes only reflect the torsional flexibility available for extension of polynucleotide chains as manifested by the crystal structures of drug-inserted oligonucleotide complexes. Intercalation proposed here could have some structural relevance elsewhere, for instance to the base-mismatched regions on the double helix and the packing of noncomplementary single strands as found in the filamentous bacteriophage Pf1.     

Fibre X-ray study of the helix-helix transitions of poly(dG-dC).poly(dG-dC).

Poly(dG-dC).poly(dG-dC) and poly(dG-m5dC).poly(dG-m5dC) present helix-helix transitions which are commonly assumed to be changes between the right-handed A- or B-DNA double helices and the left-handed Z-DNA structure. The mechanisms for such transconformations are highly improbable especially when they are supposed to be active in long polynucleotide chains organised in semicrystalline fibres. The present alternative possibility assumes that rather than the Z-DNA it is a right-handed double helix (S-DNA) which actually takes part in these form transitions. Two molecular models of this S form, in good agreement with X-ray measurements, are proposed. They present alternating C(2')-endo and C(3')-endo sugar puckering. Dihedral angles, sets of atomic co-ordinates and stereo views of the two S-DNA structures are given together with curves of calculated diffracted intensities.     

Helix-helix transitions in DNA: fibre X-ray study of the particular cases poly(dG-dC) · poly(dG-dC) and poly(dA) · 2poly(dT)

The helix-helix transitions which occur in poly(dG-dC) · poly(dG-dC) and in poly (dG-m⁵dC) · poly(dG-m⁵dC) are commonly assumed to be changes between the right-handed A- or B-DNA double helices and the left-handed Z-DNA structure. The mechanisms for such transconformations are highly improbable, especially when they are supposed to be active in long polynucleotide chains organised in semicrystalline fibres. The present alternative possibility assumes that rather than the Z-DNA it is a right-handed double helix (S-DNA) which actually takes part in these form transitions. Two molecular models of this S form, in good agreement with X-ray measurements, are proposed. They present alternating C(2′)-endo and C(3′)-endo sugar puckering like the “alternating B-DNA” put forward some years ago. Dihedral angles, sets of atomic coordinates and stereo views of the two S-DNA structures are given, together with curves of calculated diffracted intensities. Furthermore, we question the possibility of obtaining semicrystalline fibres with triple helices of poly(dA) · 2poly(dT) in a way which renders X-ray diffraction efficient. It is suggested that, up to now, only double helices of poly(dA) · poly(dT) can actually be observed by fibre X-ray diffraction measurements. Received: 30 March 1999 / Revised version: 30 June 1999 / Accepted: 30 June 1999     

Parallel DNA helices. Conformational analysis of regular poly(dG).poly(dC) helices with different variants of base binding]

We have performed a conformational analysis of DNA double helices with parallel directed backbone strands. The calculations were made for homopolymers poly(dG).poly(dC). All possible models of base binding were checked. By the potential energy optimization the dihedral angles and helices parameters of stable conformations of parallel double polynucleotides were calculated. The dependences of conformational energy on the base pair structure were studied. Possible structure of parallel helices with various nucleotide composition are discussed.     

Parallel Double Helices of DNA. Conformational Analysis of Regular Helices with the Second Order Symmetry Axis

Abstract

We have performed a conformational analysis of DNA double helices with parallel directed backbone strands connected with the second order symmetry axis being at the same time the helix axis. The calculations were made for homopolymers poly(dA) · poly(dA), poly(dC) · poly(dC), poly(dG) poly(dG), and poly(dT) · poly(dT). All possible variants of hydrogen bonding of base pairs of the same name were studied for each polymer. The maps of backbone chain geometrical existence were constructed. Conformational and helical parameters corresponding to local minima of conformational energy of “parallel” DNA helices, calculated at atom-atom approximation, were determined. The dependence of conformational energy on the base pair and on the hydrogen bond type was analysed. Two major conformational advantageous for “parallel” DNA's do not depend much on the hydrogen-bonded base pair type were indicated. One of them coincided with the conformational region typical for “antiparallel” DNA in particular for the B-form DNA Conformational energy of “parallel” DNA depends on the base pair type and for the most part is similar to the conformational energy of “antiparallel” B-DNA.     

A cooperative self-consistent microscopic theory of thermally induced melting of a repeat sequence DNA polymer

We attempt to extend the modified self-consistent phonon theory to describe thermal fluctuational base-pair opening of repeat sequence DNA polymers in the helix–coil transition region as well as in the premelting region. A microscopic base-pair open state is introduced and the effect of this open state is taken into account self-consistently in a mean field system that models the DNA polymer. Our analysis indicates the structure of this open state changes with temperature in such a manner that on average a base pair opens and unstacks with its neighbors more completely as temperature increases. We apply this theory to a homopolymer—poly(dG) · poly(dC) to evaluate the base-pair opening probability in a temperature range from 273 to 366.5 K. At 366.5 K the system undergoes cooperative melting. Our calculated base-pair opening probabilities are in general agreement with several experimental estimates at room temperature. The calculated probabilities show typical melting curve behavior at temperatures close to the observed melting temperature. The cooperative modified self-consistent phonon approximation approach becomes a viable microscopic theory of melting. © 1993 John Wiley & Sons, Inc.     

UDP-glucose dehydrogenase: substrate binding stoichiometry and affinity

Precise structural parameters of polyribonucleotides single stranded helices are determined as well as those of double stranded helices of poly 2′-O-methyl A and of poly A at neutral and acid pH. Infrared linear dichroism investigations indicate the similarity of the conformation of the sugar-phosphate backbone of these single and double stranded helices. The angles of the phosphate group for single stranded helix at neutral pH is found to be oriented at 48° for the 02P02 bisector and at about 65° for the 02–03 line to the helix axis. Similar values were found for double stranded poly A helix at acid pH. These structural parameters obtained for the first time on single stranded polynucleotide helices are proposed to be valid for other similar helical chains such as poly A segments of nuclear or messenger RNA and single stranded CCA acceptor end of transfer RNA.     

RNA double helices generated from crystal structures of double helical dinucleoside phosphates.

The dinucleoside phosphates ApU and GpC form right-handed anti-parallel double helical fragments within their crystal lattices. Using a least squares procedure, we have generated the extended double helices which these fragments represent. ApU corresponds to a double helix with 11.9 residues per turn and a pitch of 28. 1Å. The GpC double helix has 10.4 residues per turn and a pitch of 26. 9Å.     

The double-helical molecular structure of crystalline b-amylose

The crystal structure of the B-polymorph of amylose appears to be based on double-stranded helices. The individual strands are in a right-handed six-fold helical conformation repeating in 20.8 Å and are wound parallel around each other. The steric disposition of O-6 is gt. The double helices pack in a hexagonal unit-cell (a  b  18.50 Å, c (fiber repeat)  10.40 Å, γ  120°), with two helices (12 d-glucose residues) per cell. The helices are packed antiparallel and leave an open channel within a hexagonal array that is filled with water molecules. The reliability of the structure analysis is indicated by R  0.22. The structure of B-amylose is consistent with the diffraction diagrams of B-starches and accounts for the physical properties of such starches.     

A structural model for the polyadenylic acid single helix.

In the crystal, the poly(A) fragment ApApA assumes a conformation with the 5′-terminal and middle adenosines in a single helical arrangement. From the atomic co-ordinates of these two nucleotides the structure of the poly(A) single helix was derived mathematically. The helix has a pitch height of 25.4 Å, nine nucleotides per turn and the normals to the adenine bases form an angle of 66 ° with the helix axis.     

Parallel double helices of DNA. Conformational analysis of regular helices with the second order symmetry axis

We have performed a conformational analysis of DNA double helices with parallel directed backbone strands connected with the second order symmetry axis being at the same time the helix axis. The calculations were made for homopolymers poly(dA).poly(dA), poly(dC).poly(dC), poly(dG) poly(dG), and poly(dT).poly(dT). All possible variants of hydrogen bonding of base pairs of the same name were studied for each polymer. The maps of backbone chain geometrical existence were constructed. Conformational and helical parameters corresponding to local minima of conformational energy of "parallel" DNA helices, calculated at atom-atom approximation, were determined. The dependence of conformational energy on the base pair and on the hydrogen bond type was analysed. Two major conformational advantageous for "parallel" DNA's do not depend much on the hydrogen-bonded base pair type were indicated. One of them coincided with the conformational region typical for "antiparallel" DNA, in particular for the B-form DNA. Conformational energy of "parallel" DNA depends on the base pair type and for the most part is similar to the conformational energy of "antiparallel" B-DNA.     

Nucleotide sequence-dependent opening of double-stranded DNA at an electrically charged surface

It has been shown earlier that the DNA double helix is opened due to a prolonged contact of the DNA molecule with the surface of the mercury electrode. At neutral pH, the opening process is relatively slow (around 100 s), and it is limited to potentials close to -1.2 V (against SCE). The opening of the double helix has been explained by strains in the DNA molecule due to strong repulsion of the negatively charged phosphate residues from the electrode surface where the polynucleotide chain is anchored via hydrophobic bases. Interaction of the synthetic ds polynucleotides with alternating nucleotide sequences/poly(dA-dT).poly (dA-dT), poly (dA-dU).poly (dA-dU), poly (dG-dC).poly (dG-dC)/ and homopolymer pairs/poly (dA).poly (dT), poly (rA).poly (rU) and poly (dG).poly (dC)/ with the hanging mercury drop electrode has been studied. Changes in reducibility of the polynucleotides were exploited to indicate opening of the double helix. A marked difference in the behaviour was observed between polynucleotides with alternating nucleotide sequence and homopolymer pairs: opening of the double-helical structures of the former polynucleotides occurs at a very narrow potential range (less than 100 mV) (region U), while with the homopolymer pairs containing A X T or A X U pairs, the width of this region is comparable to that of natural DNA (greater than 200 mV). In contrast to natural DNA, the region U of homopolymer pairs is composed of two distinct phases. No region U was observed with poly (dG).poly (dC). In polynucleotides with alternating nucleotide sequence, the rate of opening of the double helix is strongly dependent on the electrode potential in region U, while in homopolymer pairs, this rate is less potential-dependent. It has been assumed that the difference in the behaviour between homopolymer pairs and polynucleotides with alternating nucleotide sequence is due to differences in absorbability of the two polynucleotide chains in the molecule of a homopolymer pair (resulting from different absorbability of purine and pyrimidine bases) in contrast to equal adsorbability of both chains in a polynucleotide molecule with alternating nucleotide sequence. It has been shown that the mercury electrode is a good model of biological surfaces (e.g. membranes), and that the nucleotide sequence-dependent opening (unwinding) of the DNA double helix at electrically charged surfaces may play an important role in many biological processes.     

Triple helical polynucleotidic structures: an FTIR study of the C+ .G. Ctriplet.

Triple helixes containing one homopurine poly dG or poly rG strand and two homopyrimidine poly dC or poly rC strands have been prepared and studied by FTIR spectroscopy in H2O and D2O solutions. The spectra are discussed by comparison with those of the corresponding third strands (auto associated or not) and of double stranded poly dG.poly dC and poly rG.poly rC in the same concentration range and salt conditions. The triplex formation is characterized by the study of the base-base interactions reflected by changes in the spectral domain involving the in-plane double bond vibrations of the bases. Modifications of the initial duplex conformation (A family form for poly rG.poly rC, B family form for poly dG.poly dC) when the triplex is formed have been investigated. Two spectral domains (950-800 and 1450-1350 cm-1) containing absorption bands markers of the N and S type sugar geometries have been extensively studied. The spectra of the triplexes prepared starting with a double helix containing only riboses (poly rC+.poly rG.poly rC and poly dC+.poly rG.poly rC) as well as that of poly rC+.poly dG.poly dC present exclusively markers of the North type geometry of the sugars. On the contrary in the case of the poly dC+.poly dG.poly dC triplex both N and S type sugars are shown to coexist. The FTIR spectra allow us to propose that in this case the sugars of the purine (poly dG) strand adopt the S type geometry.     

On the recognition of helical RNA by cobra venom V1 nuclease

The V1 nuclease from cobra venom preferentially hydrolyzes double helical RNA and has been used extensively for detecting RNA secondary structure. To increase the utility of this enzyme as an RNA structure probe, we have investigated its properties and substrate specificity, using assays for polynucleotide hydrolysis based on fluorescent polynucleotide derivatives. Enzymatic activity requires both Na+ and Mg2+, with optima at 100 and 0.3 mM, respectively. From the sharp decrease in enzyme activity above 100 mM Na+ we estimate that 3-4 ionic interactions between the protein and polynucleotide phosphates take place. Analysis of products remaining after extensive V1 digestion also shows that the minimum size substrate is 4-6 nucleotides long. Helical RNAs and DNAs have Michaelis constants a factor of 3-10 times lower than most single-stranded RNAs. However, poly(epsilon A) has a Michaelis constant equal to the best synthetic double helices tested and is hydrolyzed at a rate comparable to helical RNA. The major V1 cutting sites in yeast tRNAPhe have Michaelis constants lower than any synthetic polymers. These data suggest that V1 nuclease recognizes any 4-6-nucleotide segment of polynucleotide backbone with an approximately helical conformation, but does not require that the bases be paired in a helix. A few single-stranded V1 cleavage sites are known in tRNA and rRNA, and their structures are consistent with the suggested V1 recognition site.     

Crystal structure of a poly(rA) staggered zipper at acidic pH: evidence that adenine N1 protonation mediates parallel double helix formation

We have solved at 1.07 Å resolution the X-ray crystal structure of a polyriboadenylic acid (poly(rA)) parallel and continuous double helix. Fifty-nine years ago, double helices of poly(rA) were first proposed to form at acidic pH. Here, we show that 7-mer oligo(rA), i.e. rA₇, hybridizes and overlaps in all registers at pH 3.5 to form stacked double helices that span the crystal. Under these conditions, rA₇ forms well-ordered crystals, whereas rA₆ forms fragile crystalline-like structures, and rA₅, rA₈ and rA₁₁ fail to crystallize. Our findings support studies from ∼50 years ago: one showed using spectroscopic methods that duplex formation at pH 4.5 largely starts with rA₇ and begins to plateau with rA₈; another proposed a so-called ‘staggered zipper’ model in which oligo(rA) strands overlap in multiple registers to extend the helical duplex. While never shown, protonation of adenines at position N1 has been hypothesized to be critical for helix formation. Bond angles in our structure suggest that N1 is protonated on the adenines of every other rAMP−rAMP helix base pair. Our data offer new insights into poly(rA) duplex formation that may be useful in developing a pH sensor.     

Binding of CC-1065 to poly- and oligonucleotides

The binding of the antitumor agent CC-1065 to a variety of poly- and oligonucleotides was studied by electronic absorption, CD, and resistance to removal by Sephadex column chromatography. Competitive binding experiments between CC-1065 and netropsin were carried out with calf-thymus DNA, poly(dI-dC) · poly(dI-dC), poly(dI) · poly(dC), poly(rA) · poly(dT), poly(dA- dC) · poly(dG-dT), and poly(dA) · 2poly(dT). CC-1065 binds to polynucleotides by three mechanisms. In the first, CC-1065 binds only weakly, as judged by the induction of zero or very weak CD spectra and low resistance to extraction of drug from the polynucleotide by Sephadex chromatography. In the second and third mechanisms, CC-1065 binds strongly, as judged by the induction of two distinct, intense CD spectra and high resistance to extraction of drug from the polynucleotide, by Sephadex chromatography in both cases. The species bound by the second mechanism converts to that bound by the third mechanism with varying kinetics, which depend both on the base-pair sequence and composition of the polynucleotide. Competitive binding experiments with netropsin show that CC-1065 binds strongly in the minor groove of DNA by the second and third mechanisms of binding. Netropsin can displace CC-1065 that is bound by the second mechanism but not that bound by the third mechanism. CC-1065 binds preferentially to B-form duplex DNA and weakly (by the first binding mechanism) or not at all to RNA, DNA, and RNA–DNA polynucleotides which adopt the A-form conformation or to single-strand DNA. This correlation of strong binding of CC-1065 to B-form duplex DNA is consistent with x-ray data, which suggest an anomalous structure for poly(dI) · poly(rC), as compared with poly(rI) · poly(dC) (A-form) and poly(dI) · poly(dC) (B-form). The binding data indicate that poly(rA) · poly(dU) takes the B-form secondary structure like poly(rA) · poly(dT). Triple-stranded poly(dA) · 2poly(dT) and poly(dA) · 2poly(dU), which are considered to adopt the A-form conformation, bind CC-1065 strongly. Netropsin, which also shows a binding preference for B-form polynucleotides, also binds to poly(dA) · 2poly(dT) and occupies the same binding site as CC-1065. These binding studies are consistent with results of x-ray studies, which suggest that A-form triplex DNA retains some structural features of B-form DNA that are not present in A-form duplex DNA; i.e., the axial rise per nucleotide and the base tilt. Triple-stranded poly(dA) · 2poly(rU) does not bind CC-1065 strongly but has nearly the same conformation as poly(dA) · 2poly(dT) based on x-ray analysis. This suggests that the 2′-OH group of the poly(rU) strands interferes with CC-1065 binding to this polynucleotide. The same type of interference may occur for other RNA and DNA–RNA polynucleotides that bind CC-1065 weakly.     

A new D-DNA form of poly(dA-dT).poly(dA-dT): an A-DNA type structure with reversed Hoogsteen pairing

The D-DNA double helix model of poly(dA-dT).poly(dA-dT) proposed in the literature is not in accordance with some notable experimental facts and physicochemical conditions to which it is related. Thus, the fibre X-ray diffraction pattern of D-DNA obtained at a relative humidity lower than that giving the A-DNA form is singularly not taken into account when one assumes that there is only one D structure of B-DNA type. We rather suggest that there are actually two different forms of D-DNA, namely D(A) which partakes in the D-A-B transitions and D(B) associated with the D-B change of conformation. Although these two DNA structures have the same helical parameters (pitch and number of residues per turn), in agreement with X-ray data, their detailed conformations are considerably different. Whereas D(B) is indeed the structure generally defined as D-DNA, a critical analysis based on a comparison between different possible DNA double helices leads us to propose dihedral angles, a set of atomic coordinates and a stereo view of another new form of D-DNA, the D(A) structural model. It is a right-handed double helix with a dinucleotide as the repeat unit. The furanose rings are of the A-DNA type (C3' endo) and the bases are hydrogen bonded according to the reversed Hoogsteen pairing. Such a disposition renders the D(A) model unsuitable for poly(dI-dC).poly(dI-dC), the other alternating polynucleotide observed in the D(B) structure. The consistency of these two different D-DNA structures of poly(dA-dT).poly(dA-dT) with the general aspects of hydration and helix-helix transitions of DNA, as well as with the conformational variability of AT base sequences, is discussed.     

Comparison of the transmembrane helices of bovine rhodopsin in the crystal structure and the C α template based on cryo-electron microscopy maps and sequence analysis of the G protein-coupled receptors

G protein-coupled receptors (GPCR) are activated by a diverse array of extracellular signals, ranging from light to polypeptide molecules. The receptors propagate these signals intracellularly using G protein secondary messenger pathways. A common feature in the architecture of these receptors is their seven transmembrane domains. The first crystal structure of a GPCR, bovine rhodopsin, has recently been solved at 2.8 Å. We compared the seven membrane-spanning helices (TMH) from the crystal structure of bovine rhodopsin with those from the low-resolution model of bovine rhodopsin based on the cryo-electron microscopy structure of frog rhodopsin developed by Dr Joyce Baldwin. The model developed by Baldwin used a consensus sequence approach to predict the rotational position of each helix with respect to the other six helices. Superposition of the entire helix bundle of the Baldwin model with the crystal structure gave a RMS difference (RMSD) of 3.2 Å for the 198 C f atoms which suggests a high level of similarity in the arrangement of the helices. Except for TMH IV (RMSD of 4.0 Å), the position of corresponding helices within the helix bundle overlapped well. The superposition of individual helices showed that the RMSD values over 3 Å in the global superposition were largely due to one or more of the following: (i) differences in the unraveling and kinks for these helices, (ii) translation of TMH perpendicular to the membrane and (iii) rotation of helices up to 31°, except for TMH IV in which an additional contribution to the RMSD came from the aforementioned observation. As other crystal structures of GPCRs become available, a comparison with the Baldwin consensus model may reveal larger differences than those observed here.     

Geometrical principles of homomeric β‐barrels and β‐helices: Application to modeling amyloid protofilaments

Examples of homomeric β‐helices and β‐barrels have recently emerged. Here we generalize the theory for the shear number in β‐barrels to encompass β‐helices and homomeric structures. We introduce the concept of the “β‐strip,” the set of parallel or antiparallel neighboring strands, from which the whole helix can be generated giving it n‐fold rotational symmetry. In this context, the shear number is interpreted as the sum around the helix of the fixed register shift between neighboring identical β‐strips. Using this approach, we have derived relationships between helical width, pitch, angle between strand direction and helical axis, mass per length, register shift, and number of strands. The validity and unifying power of the method is demonstrated with known structures including α‐hemolysin, T4 phage spike, cylindrin, and the HET‐s(218‐289) prion. From reported dimensions measured by X‐ray fiber diffraction on amyloid fibrils, the relationships can be used to predict the register shift and the number of strands within amyloid protofilaments. This was used to construct models of transthyretin and Alzheimer β(40) amyloid protofilaments that comprise a single strip of in‐register β‐strands folded into a “β‐strip helix.” Results suggest both stabilization of an individual β‐strip helix and growth by addition of further β‐strip helices can involve the same pair of sequence segments associating with β‐sheet hydrogen bonding at the same register shift. This process would be aided by a repeat sequence. Hence, understanding how the register shift (as the distance between repeat sequences) relates to helical dimensions will be useful for nanotube design.     

On the kinetics of helix formation between complementary ribohomopolymes and deoxyribohomopolymers

The rate of double helix formation by single-stranded poly A plus poly dT, poly dA plus poly U, poly dA plus dT, poly G plus poly dC, poly dG plus poly C, and poly dG plus poly dC have been investigated and compared to rates of ribohomopolymer helix formation rates. After correction for molecular weight, comparisons of rate data at 30°C below the melting temperature of the double helix show that:

• 1 Rates of helix formation by all combinations of guanine plus cytosine homopolymers are the same.
• 1 The rate of helix formation for poly dA plus poly dT is three times faster than the rate for poly A plus poly U. Rates of formation of DNA-RNA hybrid molecules are intermediate between these two rates, but closer to the poly dA plus poly dT rate.

The effect of temperature on the rate of helix formation is interpreted in terms of a steady-state model for helix propagation. The results are consistent with a mechanism in which the formation of the second base pair is the rate-determining step.