Monoclonal antibodies specific for poly(dG) X poly(dC) and poly(dG) X poly(dm5C)

Most duplex DNAs that are in the "B" conformation are not immunogenic. One important exception is poly(dG) X poly(dC), which produces a good immune response even though, by many criteria, it adopts a conventional right-handed helix. In order to investigate what features are being recognized, monoclonal antibodies were prepared against poly(dG) X poly(dC) and the related polymer poly(dG) X poly(dm5C). Jel 72, which is an immunoglobulin G, binds only to poly(dG) X poly(dC), while Jel 68, which is an immunoglobulin M, binds approximately 10-fold more strongly to poly(dG) X poly(dm5C) than to poly(dG) X poly(dC). For both antibodies, no significant interaction could be detected with any other synthetic DNA duplexes including poly[d(Gm5C)] X poly[d(Gm5C)] in both the "B" and "Z" forms, poly[d(Tm5Cm5C)] X poly[d(GGA)], and poly[d(TCC)] X poly[d(GGA)], poly(dI) X poly(dC), or poly(dI) X poly(dm5C). The binding to poly(dG) X poly(dC) was inhibited by ethidium and by disruption of the DNA duplex, confirming that the antibodies were not recognizing single-stranded or multistranded structures. Furthermore, Jel 68 binds significantly to phage XP-12 DNA, which contains only m5C residues and will precipitate this DNA in the absence of a second antibody. The results suggest that (dG)n X (dm5C)n sequences in natural DNA exist in recognizably distinct conformations.     

Reversible helix/coil transitions of left-handed Z-DNA structures. Comparison of the thermodynamic properties of poly(dG).poly(dC), poly[d(G-C)].poly[d(G-C)], and poly(dG-m5dC).poly(dG-m5dC)

In contrast to poly(dG).poly(dC), which remains in the B-DNA conformation under all experimental conditions the polynucleotides with the strictly alternating guanine/cytosine or guanine/5'-methylcytosine sequences can change from the classical right-handed B-DNA structure to the left-handed Z-DNA structure when certain experimental conditions such as ionic strength or solvent composition are fulfilled. Up to now the investigation of the helix/coil transition of left-handed DNA structures was not possible because the transition temperature exceeds 98 degrees C. By applying moderate external pressure to the surface of the aqueous polymer solution in the sample cell the boiling point of the solvent water is shifted up the temperature scale without shifting the transition temperature, so that we can measure the helix/coil transition of the polynucleotides at all experimental conditions applied. It can thus be shown that the Z-DNA/coil transition is cooperative and reversible. The Tm is 125 degrees C for poly(dG-m5dC).poly(dG-m5dC) in 2mM Mg2+, 50mM Na+, pH 7.2 and 115 degrees c for poly[d(G-C)].poly[d(G-C)] in 3.04M Na+. The transition enthalpy per base pair was determined by the help of an adiabatic scanning microcalorimeter.     

Effect of mercuric and silver ions on cesium sulfate equilibrium buoyant densities of synthetic polydeoxyribonucleotides.

The effect of Hg2+ and Ag+ on the buoyant density (rho) of four synthetic DNA polymers, poly[d(A-T)]; poly(dA) - poly(dT); oikt[d(G-C)]; and poly(dG) - poly(dC), was investigated. The buoyant density of poly[d(A-T)] in Cs2SO4 increased dramatically after complexing with Hg2+, but little change in the buoyant density of other polymers resulted except at very high molar ratios of Hg2+/DNA-P (rf). Hg2+ raised the thermal transition temperature (Tm) of alternating polymers and lowered the Tm of homopolymers. Measurements in the preparative ultracentrifuge indicated that lowered Tm correlated with Hg2+-induced strand separation of one homopolymer [poly(dA) - poly(dT)], but strand separation was not observed with another homopolymer [poly(dG) - poly(dC)] complexed with Hg2+. When Ag+ was mixed with the polymers, the buoyant density of poly(dG) - poly(dC) increased most markedly. A substantial increase in the buoyant density of poly[d(A-T)] and a small increase in the buoyant density of poly[d(G-C)] were also observed. The Tm changes induced by Ag+ were not related in any obvious way to buoyant density changes. These findings indicate that nucleotide sequence as well as overall base composition is of importance in understanding the buoyant density changes induced by metal ions. Although these data do not allow construction of a detailed molecular model of polymer-metal ion interactions, they may be used to explain much of the behavior of naturally occurring DNA sequences, such as heterochromatic satellite sequences and 5 S and rRNA sequences, in Hg2+/Cs2SO4 and Ag+/Cs2SO4 gradients.     

Sequence-dependant polymorphism of DNA duplex in solutions

Raman spectra of six synthetic polydeoxyribonucleotide duplexes with different base sequences have been examined in aqueous solutions with different salt or nucleotide concentrations. Detailed conformational differences have been indicated between B and Z forms of poly[d(G-C)] X poly[d(G-C)], between B forms of poly[d(G-C)] X poly[d(G-C)] and poly[d(G-m5C)] X poly[d(G-m5C)], between A and B forms of poly(dG) X poly(dC), between B and "CsF" forms of poly[d(A-T)] X poly[d(A-T)], between B forms of poly[d(A-U)] X poly[d(A-U)] and poly[d(A-T)] X poly[d(A-T)], and between low- and high-salt (CsF) forms of poly(dA) X poly(dT). The Raman spectrum of calf-thymus DNA in aqueous solution was also observed and was compared with the Raman spectra of its fibers in A, B, and C forms.     

Deuteration kinetics of deoxyguanosine, deoxycytidine, and their polynucleotides by means of ultraviolet spectrophotometry

The kinetics of the hydrogen-deuterium exchange reactions of deoxyguanosine (dG), deoxycytidine (dC), double-helical poly[d(G-C)] X poly[d(G-C], and double-helical poly(dG) X poly(dC) have been examined at 20 degrees C, pH 7.0, and in low-salt (0.15 M NaCl) medium by stopped-flow ultraviolet spectrophotometry, in the spectral region of 260 to 320 nm. The rate constant was found to be 78.9 s-1 for dG-NH, 2.2 s-1 for dG-NH2, 39.3 s-1 for dC-NH2, 2.4 s-1 (fast) and 0.94 s-1 (slow) for poly[d(G-C)] X poly[d(G-C)], and 2.2 s-1 (fast) and 0.92 s-1 (slow) for poly(dG) X poly(dC). From these values, the probability of base-pair opening of the G X C containing B-form double helix is estimated to be (3 +/- 1) X 10(-3). This is much greater than what is expected from an extrapolation of the van't Hoff plot at the helix-coil transition region, i.e. at about 110 degrees C. The mechanism of these base-pair openings at 20 degrees C (as well as the mechanism of base-pair reformation) is suggested to be totally different from those in the melting temperature range.     

Preferential binding of quinolones to DNA with alternating G,C / A,T sequences: a spectroscopic study

The binding of quinolones, nalidixic acid (Nal), oxolinic acid (Oxo) with double stranded polynucleotides was undertaken by using UV-melting, UV-Vis absorption, fluorescence and CD spectroscopic techniques. The binding of Nal or Oxo to the polynucleotides under low-salt buffer conditions were determined for poly (dA).(dT), poly [d(A-T)], poly (dG).(dC), poly [d(G-C)] and E. coli DNA. The fluorescence data were analyzed using a previously established two step mechanism with two different DNA-Drug complexes [Rajeswari et al., Biochemistry 26, 6825-31 (1987)]. The first complex [DN](1) with a binding constant K(1), is formed where the interactions are 'nonspecific' and complex [DN](2) with a binding constant K(2), is formed where the interactions are "specific" which involve (additional) hydrophobic type of interactions like 'stacking' of the drug and the overall association constant is represented as K(=K(1)K(2)). The order of binding for Nal and Oxo is: poly [d(G-C)] > poly [d(A- T)] > E. coli > poly (dG).(dC) > poly (dA).(dT). Interaction of quinolones seems to be preferential in the alternating G, C or A, T stretches of DNA than those of non-alternating. Within any alternating or non-alternating in DNA sequences the G, C rich sequences have distinctly greater binding than A, T sequences. The overall association constant data (K) reveal higher binding of Oxo to DNA compared to Nal to any given polynucleotide investigated; which also explains the higher antibacterial potency of Oxo. Changes in the absorption difference spectra and in circular dichroic spectra also manifest these results. As the melting temperatures of the polynucleotides were only marginally raised in presence of the quinolone, we rule out the possibility of 'classical intercalation' of the drug. Amino group of guanine facilitates the binding of quinolones and therefore has the greater binding with the DNA. However, poly (dG).(dC) is known to exist in 'A' conformation which is not adopted by quinolones as in the case of poly (dA).(dT). Present results suggest that Nal or Oxo bind to DNA in a non-classical fashion which is partially stacking in nature.     

Study of conformation characteristics of polydeoxyribonucleotides with non-random base sequence by slow 1H----3H exchange

The rate constants of 1H----3H exchange between water and C8H-groups of purinic residues of alternating polynucleotides: poly[d(A-T)].poly[d(A-T)] (I), poly[d(G-C)].poly[d(G-C)] (II), poly[d(A-C)].poly[d(G-T)] (III) and homopolynucleotides: poly(dA).poly(dt) (IV), poly(dG).poly(dC) (V), as well as DNA E. coli, was determined in 0.15 M NaCl at 25 degrees C. The retardation of exchange observed at these conditions (compared to that of the B-form DNA) is in agreement with the model of B-alternating structure for the (I) and is attributed to the co-existence of B- and A-conformers for the (V) in solution. Absence of distinguishable differences in exchange rate constants for purinic residues of the (II), (III) and (IV) (compared to that of the B-form DNA) evidences that conformations of these polynucleotides in solution are similar to "canonical" B-form DNA and don't correlate with the model of "heteronomous" DNA which was proposed for (IV).     

Protonated polynucleotides structures - 22.CD study of the acid-base titration of poly(dG).poly(dC)

The acid-base titration (pH 8 --> pH 2.5 --> pH 8) of eleven mixing curve samples of the poly(dG) plus poly(dC) system has been performed in 0.15 M NaCl. Upon protonation, poly(dG).poly(dC) gives rise to an acid complex, in various amounts according to the origin of the sample. We have established that the hysteresis of the acid-base titration is due to the non-reversible formation of an acid complex, and the liberation of the homopolymers at the end of the acid titration and during the base titration: the homopolymer mixtures remain stable up to pH 7. A 1G:1C stoichiometry appears to be the most probable for the acid complex, a 1G:2C stoichiometry, as found in poly(C(+)).poly(I).poly(C) or poly(C(+)).poly(G).poly(C), cannot be rejected. In the course of this study, evidence has been found that the structural consequences of protonation could be similar for both double stranded poly(dG).poly(dC) and G-C rich DNA's: 1) protonation starts near pH 6, dissociation of the acid complex of poly(dG).poly(dC) and of protonated DNA take place at pH 3; 2) the CD spectrum computed for the acid polymer complex displays a positive peak at 255 nm as found in the acid spectra of DNA's; 3) double stranded poly(dG).poly(dC) embedded in triple-stranded poly(dG).poly(dG).poly(dC) should be in the A-form and appears to be prevented from the proton induced conformational change. The neutral triple stranded poly(dG).poly(dG).poly(dC) appears therefore responsible, although indirectly, for the complexity and variability of the acid titration of poly(dG).poly(dC) samples.     

A circular dichroism study of poly dG, poly dC, and poly dG:dC

We have measured the ultraviolet circular dichroism spectra of oligo d(pG)₅, poly dN AcG, poly dI, poly dC, two samples of poly dG, and four samples containing double-stranded poly dG:dC. We find that oligo d(pG)₅ and poly dG exist in self-complexed forms as well as in single-stranded forms. Unlike the self-complexed form of poly dG, the single-stranded form of poly dG can hydrogen-bond with single-stranded poly dC. We present spectral data for double-stranded poly dG:dC, which can be used to help characterize poly dG:dC preparations and which provide a basis for resolving discrepancies among other reported poly dG:dC spectra.     

Template specificities of Aclacinomycin B on the inhibition of DNA-dependent RNA synthesis in vitro

Summary The effect of Aclacinomycin B (ACM-B), an anthracycline antitumor antibiotic, on the DNA-dependent RNA synthesis using single- and double-stranded DNAs of known base content and sequence is studied. The data show that ACM-B effectively inhibits the double-stranded DNA-directed RNA synthesis with a preference of poly[d(A-T)] > poly[d(G-C)] > poly[d(I-C)]. In contrast, it has no inhibitory effect on the template function of single-stranded DNA (e.g. poly dA, poly dT, and poly dC). These results suggest that the mechanism of ACM-13 inhibition, like other anthracycline antibiotics, is by intercalation. In addition to the base specificity, there are also dramatic differences in inhibition depending on the base sequence in the DNA template. Thus, ACM-13 preferentially inhibits the alternating double-stranded copolymers over the double-stranded homopolymers; e.g. poly [d(A-T)] is inhibited to a greater extent than poly dA · poly dT and poly [d(G-C)] is inhibited more than poly dG · poly dC. Since the inhibition by ACM-13 can be totally abolished when assayed in excess amount of DNA, this result suggests that ACM-B inhibition of RNA synthesis is solely on the DNA template (which is in support of the intercalation model), and has ruled out the possibility that ACM-B may also exert an inhibitory effect on the activity of RNA polymerase per se.     

Binding of ligands to a one-dimensional heterogeneous lattice. III. Kinetic model and relaxation study of the interaction of tilorone with DNA and polynucleotides

A temperature-jump relaxation study of the interaction of tilorone with different polynucleotides and DNA has been performed. A single relaxation time, attributed to the intercalation step, has been observed in the case of poly[d(A-T)]·poly[d(A-T)], poly[d(A-C)]·poly[d(G-T)], poly[d(G-C)]·poly[d(G-C)], and poly(dG)·poly(dC). No intercalation into poly(dA)·poly(dT) occurs, and the interaction with poly(dG)·poly(dC) is different from what is observed with the other intercalating homopolymers. Refinement of the binding model is suggested from the analysis of the kinetic data. The relaxation curves obtained with DNA are well simulated based on a binding mechanism where DNA is considered a heterogeneous lattice and each type of site behaves as if it were located in the corresponding homopolymer. Poly(dA)·poly(dT) shows a unique behavior: studies of the effects of concentration and temperature indicate that tilorone acts as a probe of a process involving the polynucleotide alone. This process appears to be related to the dynamic structure of the nucleic acid and is detectable only when the bound dye is not intercalated.     

Raman spectra of single crystals of r(GCG)d(CGC) and d(CCCCGGGG) as models for A DNA, their structure transitions in aqueous solution, and comparison with double-helical poly(dG).poly(dC)

The self-complementary oligonucleotides [r(CGC)d(CGC)]2 and [d(CCCCGGGG)]2 in single-crystal and solution forms have been investigated by Raman spectroscopy. Comparison of the Raman spectra with results of single-crystal X-ray diffraction and with data from polynucleotides permits the identification of a number of Raman frequencies diagnostic of the A-helix structure for GC sequences. The guanine ring frequency characteristic of C3'-endo pucker and anti base orientation is assigned at 668 +/- 2 cm-1 for both dG and rG residues of the DNA/RNA hybrid [r(GCG)d(CGC)]2. The A-helix backbone of crystalline [r(GCG)d(CGC)]2 is altered slightly in the aqueous structure, consistent with the conversion of at least two residues to the C2'-endo/anti conformation. For crystalline [d(CCCCGGGG)]2, the Raman and X-ray data indicate nucleosides of alternating 2'-endo-3'-endo pucker sandwiched between terminal and penultimate pairs of C3'-endo pucker. The A-A-B-A-B-A-A-A backbone of the crystalline octamer is converted completely to a B-DNA fragment in aqueous solution with Raman markers characteristic of C2'-endo/anti-G (682 +/- 2) and the B backbone (826 +/- 2 cm-1). In the case of poly(dG).poly(dC), considerable structural variability is detected. A 4% solution of the duplex is largely A DNA, but a 2% solution is predominantly B DNA. On the other hand, an oriented fiber drawn at 75% relative humidity reveals Raman markers characteristic of both A DNA and a modified B DNA, not unlike the [d-(CCCCGGGG)]2 crystal. A comparison of Raman and CD spectra of the aqueous [d(CCCCGGGG)]2 and poly(dG).poly(dC) structures suggests the need for caution in the interpretation of CD data from G clusters in DNA.     

Protonated polynucleotide structures, 20. Interaction between poly(dG)-poly(dC) and poly(rC).1.

A study of the interaction between poly(dG)-poly(dC) and poly(rC) demonstrates that, at neutral pH and high ionic strength, there is replacement of the dC strand by poly(rC). At acid pH, formation of a triple-stranded complex which equally may involve the replacement phenomenon is observed. There is no evidence for interaction at neutral pH between poly(dG)-poly(dC) and oligo(rC), while a three-stranded complex is formed at acid pH. These data are consistent with the studies of comparative stabilities of double stranded deoxy or ribo polymers and deoxy-ribo hybrids.     

Factor D is a selective single-stranded oligodeoxythymidine binding protein.

Factor D, a protein purified from rabbit liver that selectively enhances traversal of template oligodeoxythymidine tracts by diverse DNA polymerases, was examined for the sequence specificity of its binding to DNA. Terminally [32P]-labeled oligomers with the sequence 5'-d[AATTC(N)16G]-3', N being dT, dA, dG, or dC, were interacted with purified factor D and examined for the formation of protein-DNA complexes that exhibit retarded electrophoretic mobility under nondenaturing conditions. Whereas significant binding of factor D to 5'-d[AATTC(T)16G]-3' is detected, there is no discernable association between this protein and oligomers that contain 16 contiguous moieties of dG, dA, or dC. Furthermore, factor D does not form detectable complexes with the duplexes oligo(dA).oligo(dT) or poly(dA).poly(dT). The preferential interaction of factor D with single-stranded poly(dT) is confirmed by experiments in which the polymerase-enhancing activity of this protein is protected by poly(dT) against heat inactivation two- and four-fold more efficiently than by poly(dA) or poly(dA).poly(dT), respectively.     

Detection of triple-helix related structures adopted by poly(dG)-poly(dC) sequences in supercoiled plasmid DNA.

Negative superhelical strain induces the poly(dG)-poly(dC) sequence to adopt two totally different types of triple-helices, either a dG.dG.dC triplex in the presence of Mg(+)+ at both neutral and acidic pHs or a protonated dC+.dG.dC triplex in the absence of Mg(+)+ ions at acidic pH (1). To examine whether there are still other types of non-B DNA structures formed by the same sequence, we constructed supercoiled plasmid DNAs harboring varying lengths of the poly(dG) tract, and the structures adopted by each supercoiled plasmid DNA were studied with a chemical probe, chloroacetaldehyde. The potential of a poly(dG)-poly(dC) sequence to adopt non-B DNA structures depends critically on the length of the tract. Furthermore, in the presence of Mg(+)+ and at a mildly acidic pH, in addition to the expected dG.dG.dC triplex detected for the poly(dG) tracts of 14 to 30 base pairs (bp), new structures were also detected for the tracts longer than 35 bp. The structure formed by a poly(dG) tract of 45 bp revealed chemical reaction patterns consistent with a dG.dG.dC triplex and protonated dC+.dG.dC triple-helices fused together. This structure lacks single-stranded stretches typical of intramolecular triplexes.     

Rabbit liver factor D, a poly(thymidine) template stimulatory protein of DNA polymerases: purification and characterization

Factor D, a DNA binding protein that enhances the activities of diverse DNA polymerases with a common restricted set of templates, was initially characterized in mouse liver but has resisted extensive purification. In this paper, we report that a similar stimulatory activity can be obtained in highly purified form from nuclei of rabbit hepatocytes. The rabbit liver protein increases the rates at which several DNA polymerases copy sparsely primed natural DNA templates and primed synthetic poly(dT), but it has no effect on the rates of copying of activated DNA or of poly(dG), poly(dA), and poly(dC). Direct binding of the purified stimulatory protein to an oligomer that contains a (dT)16 base stretch is visualized by retardation of the nucleoprotein complex on nondenaturing electrophoretograms. In the presence of the enhancing factor, Michaelis constants, Km, of responsive polymerase for singly primed bacteriophage M13 DNA and for poly(dT), but not for poly(dA), are decreased. Product analysis of M13 DNA primer extension indicates that the rabbit factor augments the apparent processivity of DNA polymerase by decreasing the extent of enzyme pausing at a tract of four consecutive thymidine residues in the template. Gel filtration of the native stimulatory protein yields an apparent relative molecular size of 58 +/- 2 kilodaltons. Stimulatory activity is readily inactivated by heat or by trypsin digestion, but it is resistant to micrococcal nuclease, N-ethyl-maleimide, or calcium ions.     

Inhibition of RNA-directed DNA polymerase by aurintricarboxylic acid.

Commercial-grade aurintricarboxylic acid (ATA) inhibits poly(A), poly(C) and viral RNA-directed DNA synthesis by detergent-disrupted virions of Moloney murine leukemia virus. Paper chromatography of crude ATA yields two active components, which appear to behave identically, and at least two inactive components. The concentration of ATA needed to inhibit polymerase activity is proportional to the concentration of viral protein. The inhibition is neither attributable to contaminating heavy metal ions in the ATA preparation nor to chelation by ATA of Mn2+ or Zn2+, the necessary co-factors. Inhibition of the polymerase reaction by ATA greatly increases the Km for the primer [oligo(T)/oligo(dG)], while it only slightly lowers the Vmax and does not affect the Km's for the template [poly(A)/poly(C)] or the substrate (TTP/dGTP). Thus, ATA seems to reduce specifically the affinity of the polymerase for the DNA primer molecule.