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).     

DNA with adenine tracts contains poly(dA).poly(dT) conformational features in solution.

The conformation of DNA's with adenine-thymine tracts exhibiting retardation in electrophoretic migration and considered as curved were investigated in solution by CD and RAMAN spectroscopy. The following curved multimers with adenine tracts but of different flanking sequences d(CA5TGCC)n, d(TCTCTA6TATATA5)n, d(GA4T4C)n yield CD spectroscopic features indicating a non-B structure of the dA.dT tract with similarities to polyd(A).polyd(T). We suggest that adenine-thymine bases in these multimers contain some of the distinctive conformational features of poly(A).polyd(T) probably with large propeller twist found by NMR (Behling and Kearns, 1987) and by X-ray diffraction on oligonucleotides containing a tract of adenines (Nelson et al. 1987, Coll et al; 1987; DiGabriele et al. 1989). Some elements of distinctive CD features of the contiguous adenines run are also observed in the straight multi-9-mer d(CA5GCC)n which lacks in-phase relation to the helical repeat. Despite the presence of the TpA step in the straight multimer d(GT4A4)n, the altered dA.dT conformation is not completely destroyed. Interruption of adenine tract by a guanine in d(CAAGAATGCC)n leads to a B-like conformation and to a normal electrophoretic mobility. The Raman spectra reveal a rearrangement of the sugar-phosphate backbone of dA.dT tract in the multimer d(CA5TGCC)n with respect to that of polydA.polydT. This is reflected in the presence of an unique Raman band associated to C2'-endo sugar with a predominant contribution of C1'-exo puckering which is exhibited by the multimer whereas two distinct Raman bands characterize poly(dA).poly(dT) backbone conformation.     

The unusual conformation adopted by the adenine tracts in kinetoplast DNA

To determine the structural features responsible for the curvature of kinetoplast DNA, we studied 13 adenine tracts in Crithidia fasciculata kinetoplast DNA. The structures of the A tracts were analyzed by cutting the DNA with hydroxyl radical. Reactivity of hydroxyl radical toward the DNA backbone progressively decreased in the 5'----3' direction of each A tract. The cutting pattern of the T-rich strand was offset by 1 or 2 bp from the pattern on the A-rich strand. An A tract in a restriction fragment from plasmid pBR322 had the same cutting pattern as the kinetoplast A tracts. We interpret these experiments to show that in A tracts the width of the minor groove decreases smoothly from the 5'----3' end of the A tract.     

Complete disproportionation of duplex poly(dT)*poly(dA) into triplex poly(dT)*poly(dA)*poly(dT) and poly(dA) by coralyne

Coralyne is a small crescent-shaped molecule known to intercalate duplex and triplex DNA. We report that coralyne can cause the complete and irreversible disproportionation of duplex poly(dT)·poly(dA). That is, coralyne causes the strands of duplex poly(dT)·poly(dA) to repartition into equal molar equivalents of triplex poly(dT)·poly(dA)·poly(dT) and poly(dA). Poly(dT)·poly(dA) will remain as a duplex for months after the addition of coralyne, if the sample is maintained at 4°C. However, disproportionation readily occurs upon heating above 35°C and is not reversed by subsequent cooling. A titration of poly(dT)·poly(dA) with coralyne reveals that disproportionation is favored by as little as one molar equivalent of coralyne per eight base pairs of initial duplex. We have also found that poly(dA) forms a self-structure in the presence of coralyne with a melting temperature of 47°C, for the conditions of our study. This poly(dA) self-structure binds coralyne with an affinity that is comparable with that of triplex poly(dT)·poly(dA)·poly(dT). A Job plot analysis reveals that the maximum level of poly(dA) self-structure intercalation is 0.25 coralyne molecules per adenine base. This conforms to the nearest neighbor exclusion principle for a poly(dA) duplex structure with A·A base pairs. We propose that duplex disproportionation by coralyne is promoted by both the triplex and the poly(dA) self-structure having binding constants for coralyne that are greater than that of duplex poly(dT)·poly(dA).     

Poly(dA).poly(dT) exists in an unusual conformation under physiological conditions: propidium binding to poly(dA).poly(dT) and poly[d(A-T)].poly[d(A-T)]

The binding of propidium to poly(dA).poly(dT) [poly(dA.dT)] and to poly[d(A-T)].poly[d(A-T)] [poly[d(A-T)2]] has been compared under a variety of solution conditions by viscometric titrations, binding studies, and kinetic experiments. The binding of propidium to poly[d(A-T)2] is quite similar to its binding to calf thymus deoxyribonucleic acid (DNA). The interaction with poly(dA.dT), however, is quite unusual. The viscosity of a poly(dA.dT) solution first decreases and then increases in a titration with propidium at 18 degrees C. The viscosity of poly[d(A-T)2] shows no decrease in a similar titration. Scatchard plots for the interaction of propidium with poly(dA.dT) show the classical upward curvature for positive cooperativity. The curvature decreases as the temperature is increased in binding experiments. A van't Hoff plot of the observed binding constants yields an apparent positive enthalpy of approximately +6 kcal/mol for the propidium-poly(dA.dT) interaction. Propidium binding to poly[d(A-T)2] shows no evidence for positive cooperativity, and the enthalpy change for the reaction is approximately -9 kcal/mol. Both the magnitude of the dissociation constants and the effects of ionic strength are quite similar for the dissociation of propidium from poly(dA-T)2] and from poly[d(A-T)2], suggesting that the intercalated states are similar for the two complexes. The observed association reactions, under pseudo-first-order conditions, are quite different. Plots of the observed pseudo-first-order association rate constant vs. polymer concentration have much larger slopes for propidium binding to poly[d(A-T)2] than to poly(dA.dT).(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure of poly (dT).poly (dA).poly (dT)

The molecular structure of poly (dT).poly (dA).poly (dT) has been determined and refined using the continuous x-ray intensity data on layer lines in the diffraction pattern obtained from an oriented fiber of the DNA. The final R-value for the preferred structure is 0.29 significantly lower than that for plausible alternatives. The molecule forms a 12-fold right-handed triple-helix of pitch 38.4 A and each base triplet is stabilized by a set of four Crick-Watson-Hoogsteen hydrogen bonds. The deoxyribose rings in all the three strands have C2'-endo conformations. The grooveless cylindrical shape of the triple-helix is consistent with the lack of lateral organization in the fiber.     

High pressure fourier transform infrared spectroscopy of poly(dA)poly(dT), poly(dA) and poly(dT)

The effect of hydrostatic pressure upon the DNA duplex, poly(dA)poly(dT), and its component single strands, poly(dA) and poly(dT) has been studied by fourier-transform infrared spectroscopy (FT-IR). The spectral data indicate that at 28 degrees C and pressures up to 12 kbar (1200 MPa) all three polymers retain the B conformation. Pressure causes the band at 967 cm(-1), arising from water-deoxyribose interactions, to shift to higher frequencies, a result consistent with increased hydration at elevated pressures. A larger pressure-induced frequency shift in this band is observed in the single stranded polymers than in the double stranded molecule, suggesting that the effect of pressure on the hydration of single strands may be greater than upon a double stranded complex. A pressure-dependent hypochromicity in the bands attributed to base stacking indicates that pressure facilitates the base stacking in the three polymers, in agreement with previous assessments of the importance of stacking in the stabilization of DNA secondary structure at ambient and high pressures.     

Nucleosomes will not form on double-stranded RNa or over poly(dA).poly(dT) tracts in recombinant DNA.

We have been unable to "force" double-stranded RNA to fold into nucleosome-like structures using several different histone-RNA "reconstitution" procedures. Even if the histones are first stabilized in octameric form by dimethylsuberimidate cross-linking they are still unable to form specific complexes with the RNA. Moreover double-stranded RNA is unable to induce histones to assemble into octamers although we confirm that the non-nucleic acid homopolymer, polyglutamic acid, has this ability. We have also determined, using pyrimidine tract analysis, that nucleosomes will not form over a sufficiently long segment of poly(dA).poly(dT) in a recombinant DNA molecule. Thus nucleosomes cannot fold DNA containing an 80 base pair poly(dA).poly(dT) segment but a 20 base pair segment can be accommodated in nucleosomes fairly well. Segments of intermediate length can be accommodated but are clearly selected against. Poly(dA).poly(dT) differs only slightly from natural DNA in helix structure. Therefore either this homopolymer resists folding, or nucleosomes are very exacting in the nucleic acid steroid parameters they will tolerate. Such constraints may be relevant to nucleosome positioning in chromatin.     

Stability of triple-helical poly(dT)-poly(dA)-poly(dT) DNA with counterions.

Structural conformation of triple-helical poly(dT)-poly(dA)-poly(dT) has been a very controversial issue recently. Earlier investigations, based on fiber diffraction data and molecular modeling, indicated an A-form conformation with C'3-endo sugar pucker. On the other hand, Raman, solution infrared spectral, and NMR studies show a B-form structure with C'2-endo sugars. In accordance with these experimental results, a theoretical model with B-form, C'2-endo sugars was proposed in 1993. In the present work we investigate the dynamics and stability of the two conformations within the effective local field approach applied to the normal mode calculations for the system. The presence of counterions was explicitly taken into account. Stable equilibrium positions for the counterions were calculated by analyzing the normal mode dynamics and free energy of the system. The breathing modes of the triple helix are shifted to higher frequencies over those of the double helix by 4-16 cm-1. The characteristic marker band for the B conformation at 835 cm-1 is split up into two marker bands at 830 and 835 cm-1. A detailed comparison of the normal modes and the free energies indicates that the B-form structure, with C'2-endo sugar pucker, is more stable than the A-form structure. The normal modes and the corresponding dipole moments are found to be in close agreement with recent spectroscopic findings.     

Four-stranded DNA. Conformational analysis of regular spirals of poly(dT).poly(dA).poly(dA).poly(dT) with different base binding variations

Conformational analysis of four stranded DNA helices poly(dT).poly(dA).poly(dA).poly(dT) with parallel arrangement of the identical sugar-phosphate chains connected by twofold symmetry has been performed. All possible models of symmetrical base binding were checked. By the potential energy optimization the dihedral angles and helices parameters of stable conformations of four stranded polynucleotides were calculated. The dependences of conformational energy on the base complex structure and mutual orientation of the poly(dA).and poly(dT) chains were studied. Possible biological functions of four stranded helices are discussed.     

Four-stranded DNA helices: conformational analysis of regular poly(dT).poly(dA).poly(dA).poly(dT) helices with various types of base binding

The paper presents results obtained in conformational analysis of homopolymeric four-stranded poly(dT).poly(dA).poly(dA).poly(dT) DNA helices in which the pairs of strands with identical bases are parallel and have a two-fold symmetry axis. All possible models of base binding to yield a symmetric complex have been considered. The dihedral angles of sugar-phosphate backbones and helix parameters, which are consistent with the minima of conformational energy for four-stranded DNAs, have been determined using the results of optimization of conformational energy calculated at atom-atom approximation. Potential energy is shown to depend on the structure of base complexes and on the mutual orientation of unlike strands. Possible biological functions of four-stranded helices are discussed.     

Inhibition of DNA replication by berenil in plasmids containing Poly(dA)poly(dT) sequences

The effect of berenil on plasmid DNA replication was studied on pBR322-derived plasmids containing poly(dA)poly(dT) sequences. In comparison to the parental plasmid pBR322, plasmid pKH47 harboring 100 bp of poly(dA)poly(dT) at the PvuII site showed a decrease in plasmid yield in the presence of berenil. This effect was also observed in pVL26, a related plasmid in which the location of the poly(dA)poly(dT) region had been shifted to the EcoRV site in pBR322. [(3)H]Thymidine incorporation experiments indicated that DNA synthesis may be affected in these plasmids in the presence of the drug. Bromodeoxyuridine incorporation experiments coupled to Cs(2)SO(4) equilibrium density gradient centrifugation indicated that the lower plasmid yield was due to an inhibition of DNA replication by berenil. We have also found that berenil induces DNA degradation in plasmids containing the homopolymer. Our studies strongly suggest that the effect of berenil on plasmid replication and DNA stability results from its binding to the poly(dA)poly(dT) region present in these plasmids. Moreover, we have found a correlation between the position of the poly(dA)poly(dT) region and this inhibitory effect. Thus, plasmid pKH47, containing the poly(dA)poly(dT) region most proximal to the origin of pBR322 replication, was most severely affected.     

A unique deletion in pBR322 DNA caused by insertion of poly(dA).poly(dT)

A unique deletion covering around 43% of the pBR322 genome was found after attempting to insert 100 or 200 bp poly(dA).poly(dT) into the EcoRV site of pBR322 DNA. This result was not observed if an equivalent size heterologous DNA or a larger poly(dA).poly(dT) fragment of 10-20,000 bp was introduced at the same site. DNA sequencing analysis at the junctions suggests that a specific intramolecular pairing may be involved in the formation of this deletion mutant.     

The structure of poly(dA).poly(dT) as revealed by an X-ray fibre diffraction

X-ray diffraction in fibres revealed that the calcium salt of poly(dA).poly(dT) is a 10-fold double helix with a pitch of 3.23 nm. The opposite sugar-phosphate chains in the refined model are characterized by a complete conformational equivalence and contain sugars in a conformation close to C2'-endo. As a result a new model of the sodium salt of poly(dA).poly(dT) has been constructed, which is different from the Heteronomous DNA proposed earlier (S. Arnott et al., Nucl. Acids Res. 11, 4141 (1983)). The new model of Na-poly(dA).poly(dT) has conformationally similar opposite chains; it is a structure of the B-type, rather like that of Ca-poly(dA).poly(dT).     

Distamycin paradoxically stimulates the copying of oligo(dA).poly(dT) by DNA polymerases

Distamycin A, a polypeptide antibiotic, binds to dA.dT-rich regions in the minor groove of B-DNA. By virtue of its nonintercalating binding, distamycin acts as a potent inhibitor of the synthesis of DNA both in vivo and in vitro. Here we report that distamycin paradoxically stimulates Escherichia coli DNA polymerase I (pol I), its large (Klenow) fragment, and bacteriophage T4 DNA polymerase to copy oligo(dA).poly(dT) in vitro. It is found that distamycin increases the maximum velocity (Vmax) of the extension of the oligo(dA) primer by pol I without affecting the Michaelis constant (Km) of the primer. Gel electrophoresis of the extended primer indicates that the antibiotic specifically increases the rate of addition of the first three dAMP residues. Lastly, in the presence of both distamycin and the oligo(dT)-binding protein factor D, which increases the processivity of pol I, a synergistic stimulation of polymerization is attained. Taken together, these results suggest that distamycin stimulates synthesis by increasing the rate of initiation of oligo(dA) extension. The stimulatory effect of distamycin is inversely related to the stability of the primer-template complex. Thus, maximum stimulation is exerted at elevated temperatures and with shorter oligo(dA) primers. That distamycin increases the thermal stability of [32P](dA)9.poly(dT) is directly demonstrated by electrophoretic separation of the hybrid from dissociated [32P](dA)9 primer. It is proposed that by binding to the short primer-template duplex, distamycin stabilizes the oligo(dA).poly(dT) complex and, therefore, increases the rate of productive initiations of synthesis at the primer terminus.     

Evidence for long poly(dA).poly(dT) tracts in D. discoideum DNA at high frequencies and their preferential avoidance of nucleosomal DNA core regions

The eukaryote, Dictyostelium discoideum, has one of the most (A+T) rich genomes studied to date. Isolated nuclear D. discoideum DNA (AX3 strain) was used to qualitatively determine the frequency and length distribution of long (dA).(dT) homopolymer tracts in this genome, in comparison to the less (A+T) rich calf thymus and Schistosoma mansoni DNAs that had few observable long tracts. These experimental data accurately reflect the significantly elevated frequencies of long tracts found computationally within the D. discoideum intron and flanking sequences, but not exons. PCR amplification of long (dA).(dT) homopolymer tract containing sequences was carried out. Then experimental biotinylated (dT)18 probe hybridization to the PCR amplified DNA showed that the long (dA).(dT) homopolymer tracts were enriched in D. discoideum sequences only hundreds of base pair in length, under conditions where no equivalent hybridization was observed to S. mansoni DNA or calf DNA sequences. Similar probe hybridization to DNA isolated following micrococcal nuclease digestion of D. discoideum chromatin demonstrated that long (dA).(dT) homopolymer tracts were more highly enriched in nucleosomal DNA lengths that included the internucleosomal linker as compared to shorter linker free mononucleosomal lengths. This observation is in agreement with the frequency of tract spacing results calculated from GenBank sequence data. These frequency data indicate that adjacent long tracts plus the intervening spacer DNA are found at peak lengths (average 42 bp), exactly characteristic of the internucleosomal spacer region of D. discoideum chromatin and are in sufficient number to be found in nearly half of all nucleosomes. Compared to shuffled tract sequence controls, these lengths of adjacent long tracts plus the intervening spacer DNA were found to be significantly enriched. Lesser enrichments are observed at lengths corresponding to adjacent tracts being separated by nucleosomal core length DNA sequences (145-185 bp). These data strongly suggest that adjacent long tracts occur spaced at selected lengths so as to avoid the central core regions of nucleosomes and instead are found localized within internucleosomal DNA linker and core edge regions in D. discoideum chromatin.     

A unique deletion in pBR322 DNA caused by insertion of poly(dA) · poly(dT)

A unique deletion covering around 43% of the pBR322 genome was found after attempting to insert 100 or 200 bp poly(dA) · poly(dT) into the EcoRV site of pBR322 DNA. This result was not observed if an equivalent size heterologous DNA or a larger poly(dA) · poly(dT) fragment of 10–20,000 bp was introduced at the same site. DNA sequencing analysis at the junctions suggests that a specific intramolecular pairing may be involved in the formation of this deletion mutant.