500-MHz 1H NMR study of poly(dG).poly(dC) in solution using one-dimensional nuclear Overhauser effect

Secondary structures of poly(dG).poly(dC) and poly(dG).poly(dm5C) in solution are determined by nuclear Overhauser effect (NOE) measurements on GH8-deuterated and -nondeuterated DNAs with low presaturation pulse lengths (10-25 ms) and low-power and prolonged accumulations in the range of 50,000-72,000 scans. Under these conditions, the NOE difference spectra were free from diffusion. Primary NOEs between base protons GH8/CH6 and sugar protons H1', H2'/H2', and H3' suggest that in poly(dG).poly(dC) both guanine and cytosine nucleotides adopt a C3'-endo, low anti X = 200-220 degrees conformation. Computer modeling of the NOE data enable identification for the first time, in terms of the geometry of the nucleotide repeat, handedness, and helix geometry, of the structure of poly(dG).poly(dC) to be the A form, and the derived structure for the polymer duplex is very close to the single crystal structure of the double-helical d-GGGGCCCC [McCall, M., Brown, T., & Kennard, O. (1985) J. Mol. Biol. 183, 385-396]. Similar nuclear Overhauser effect data on poly(dG).poly(dm5C) revealed that G and m5C adopt a C2'endo, anti X = 240-260 degrees conformation, which indicates that this DNA exhibits the B form in solution. In summary, the results presented in this paper demonstrate that methylation of cytosines in poly(dG).poly(dC) causes A----B transition in the molecule.     

A monoclonal antibody specific for the duplex DNA poly[d(TC)].poly[d(GA)]

Although most duplex DNAs are not immunogenic some synthetic DNAs such as poly[d(Tm5C)].poly[d(GA)] are weakly immunogenic allowing the production of monoclonal antibodies. The specificity of one of these antibodies, Jel 172, was investigated in detail by a competitive solid-phase radioimmune assay. Jel 172 bound well to poly[d(TC)].poly[d(GA)] but not to other duplex DNAs such as poly[d(TTC)].poly[d(GAA)] and poly[d(TCC)].poly[d(GGA)]. The binding to poly[d(Br5UC)].poly[d(GA)] was enhanced while that to poly[d(TC)].poly[d(IA)] was decreased compared to poly[d(TC)].poly[D(GA)]. Thus, not only is the antibody very specific for a sequence of duplex DNA but it also appears to recognize functional groups in both grooves of the helix.     

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.     

Thermodynamic analysis of monoclonal antibody binding to duplex DNA.

A technique based on fluorescence polarization (anisotropy) was used to measure the binding of antibodies to DNA under a variety of conditions. Fluorescein-labeled duplexes of 20 bp in length were employed as the standard because they are stable even at low ionic strength yet sufficiently short so that both arms of an IgG cannot bind to the same duplex. IgG Jel 274 binds duplexes in preference to single-stranded DNA; in 80 mM NaCl Kobs for (dG)20.(dC)20 is 4.1x10(7) M-1 compared with 6.4x10(5) M-1 for d(A5C10A5). There is little sequence specificity, but the interaction is very dependent on ionic strength. From plots of log Kobs against log[Na+] it was deduced that five or six ion pairs are involved in complex formation. At low ionic strength,Kobs is independent of temperature and complex formation is entropy driven with DeltaH degrees obs and DeltaC degrees p,obs both zero. In contrast, in 80 mM NaCl DeltaC degrees p,obs is -630 and -580 cal mol-1K-1 for [d(TG)]10.[d(CA)]10 and (dG)20.(dC)20 respectively. IgG Jel 241 also binds more tightly to duplexes than single-stranded DNA, but sequence preferences were apparent. The values for Kobs to [d(AT)]20 and [d(GC)]20 are 2.7x10(8) and 1.3x10(8) M-1 respectively compared with 5.7x10(6) M-1 for both (dA)20. (dT)20 and (dG)20.(dC)20. As with Jel 274, the binding of Jel 241 is very dependent on ionic strength and four or five ionic bonds are involved in complex formation with all the duplex DNAs which were tested. DeltaC degrees p,obs for Jel 241 binding to [d(AT)]20 was negative (-87 cal mol-1K-1) in 80 mM NaCl but was zero at high ionic strength (130 mM NaCl). Therefore, for duplex-specific DNA binding antibodies DeltaC degrees p,obs is dependent on [Na+] and a large negative value does not correlate with sequence-specific interactions.     

Variations in duplex DNA conformation detected by the binding of monoclonal autoimmune antibodies.

Four monoclonal antibodies (Jel 229, 239, 241, 242) which bound to duplex DNA were prepared from two autoimmune female NZB/NZW mice. Their binding to various nucleic acids was investigated by a competitive solid phase radioimmune assay which allows the estimation of relative binding constants. None of the antibodies showed any consistent variation of binding constant with base composition and thus they must recognize features of the DNA backbone. Jel 241 binds across the major groove but the interaction with poly(pyrimidine) X poly(purine) DNAs was barely detectable. This antibody appears to recognize the "alternating-B" conformation which is promoted by methylation of pyrimidines in alternating sequences. The other three antibodies bind in the minor groove. In particular, for Jel 229 the preferred antigen was poly(dG) X poly(dC) with only weak binding to poly(dA) X poly(dT). This suggests a requirement for a wide minor groove. Thus autoimmune antibodies provide examples of "analogue" recognition and can be used to detect structural variations in the grooves of duplex DNA.     

Poly(dG)-poly(dC) DNA appears shorter than poly(dA)-poly(dT) and possibly adopts an A-related conformation on a mica surface under ambient conditions

Three types of DNA: approximately 2700 bp polydeoxyguanylic olydeoxycytidylic acid [poly(dG)-poly(dC)], approximately 2700 bp polydeoxyadenylic polydeoxythymidylic acid [poly(dA)-poly(dT)] and 2686 bp linear plasmid pUC19 were deposited on a mica surface and imaged by atomic force microscopy. Contour length measurements show that the average length of poly(dG)-poly(dC) is approximately 30% shorter than that of poly(dA)-poly(dT) and the plasmid. This led us to suggest that individual poly(dG)-poly(dC) molecules are immobilized on mica under ambient conditions in a form which is likely related to the A-form of DNA in contrast to poly(dA)-poly(dT) and random sequence DNA which are immobilized in a form that is related to the DNA B-form.     

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.     

Factor C from rabbit liver. A new poly(dC) and poly[d(G-C)] template-selective stimulatory protein of DNA polymerases

We have undertaken a search for mammalian DNA-binding proteins that enhance the activity of DNA polymerases in a template sequence-specific fashion. In this paper, we report the extensive purification and characterization of a new DNA-binding protein from rabbit liver that selectively stimulates DNA polymerases to copy synthetic poly[d(G-C)] and the poly(dC) strand of poly(dC).poly(dG) as well as single-stranded natural DNA that contains stretches of oligo(dC). The enhancing protein, a polypeptide of 65 kDa designated factor C, stimulates the copying of the two synthetic templates by Escherichia coli DNA polymerase I, Micrococcus luteus polymerase, and eukaryotic DNA polymerases alpha and beta, but not by avian myeloblastosis virus polymerase. Factor C, however, does not affect utilization by these polymerases of the poly(dG) strand of poly(dC).poly(dG), of poly(dC) primed by oligo(dG), or of poly(dA).poly(dT) and poly[d(A-T)]. With polymerase I, Michaelis constants (Km) of poly[d(G-C)] and of the poly(dC) strand of poly(dC).poly(dG) are decreased by factor C 37- and 4.7-fold, respectively, whereas maximum velocity (Vmax) remains unchanged. By contrast, neither the Km value of the poly(dG) strand of poly(dC).poly(dG) nor the Vmax value with this template is altered by factor C. Rates of copying of activated DNA, denatured DNA, or singly primed M13 DNA are not affected significantly by factor C. However, primer extension analysis of the copying of recombinant M13N4 DNA that contains runs of oligo(dC) within an inserted thymidine kinase gene shows that factor C increases processivity by specifically augmenting the efficiency at which polymerase I traverses the oligo(dC) stretches. Direct binding of factor C to denatured DNA is indicated by retention of the protein-DNA complex on columns of DEAE-cellulose. Binding of factor C to poly[d(G-C)] is demonstrated by the specific adsorption of the enhancing protein to columns of poly[d(G-C)]-Sepharose. We propose that by binding to poly[d(G-C)] and to poly(dC).poly(dG), factor C enables tighter binding of some DNA polymerases to these templates and facilitates enzymatic activity.     

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.     

The isolation of duplex DNA fragments containing (dG.dC) clusters by chromatography on poly(rC)-Sephadex.

Duplex DNA containing oligo(dG.dC)-rich clusters can be isolated by specific binding to poly(rC)-Sephadex. This binding, probably mediated by the formation of an oligo(dG.dC)rC+ triple helix, is optimal at pH 5 in 50% formamide, 2 M LiCl; the bound DNA is recovered by elution at pH 7.5. Using this method we find that the viral DNAs PM2, lambda and SV40 contain at least 1, 1 and 2 sites for binding to poly(rC)-Sephadex, respectively. These binding sites have been mapped in the case of SV40; the binding sites can in turn be used for physical mapping studies of DNAs containing (dG.dC) clusters. Inspection of the sequence of the bound fragments of SV40 DNA shows that a (dG.dC)6-7 tract is required for the binding of duplex DNA to poly(rC)-Sephadex. Although about 60% of rabbit DNA cleaved with restriction endonuclease KpnI binds to poly(rC)-Sephadex, no binding is observed for the 5.1 kb DNA fragment generated by KpnI digestion, which contains the rabbit beta-globin gene. This indicates that oligo(dG.dC) clusters are not found close to the rabbit beta-globin gene.     

Endonuclease G: a (dG)n X (dC)n-specific DNase from higher eukaryotes.

An endonuclease activity (termed endonuclease G) that selectively cleaves DNA at (dG)n X (dC)n tracts has been partially purified from immature chicken erythrocyte nuclei. Sites where n greater than or equal to 9 are cleaved in a manner that resembles types II and III restriction nucleases. The nicking rate of the G-strand is 4- to 10-fold higher than that of the C-strand depending on the length of the (dG)n X (dC)n tract and/or nucleotide composition of the flanking sequences. Endonuclease G hydrolyzes (dG)24 X (dC)24 of supercoiled DNA in a bimodal way every 9-11 nucleotides, the maxima in one strand corresponding to minima in the opposite, suggesting that it binds preferentially to one side of the double helix. The nuclease produces 5' phosphomonoester ends and its activity is dependent on Mg2+ or Mn2+. The wide distribution and high relative activity of endonuclease G in a variety of tissues and species argues for a general role of the enzyme. The striking correlation between genetic instability and poly(dG) X poly(dC) tracts in DNA suggests that these sequences and endonuclease G are involved in recombination processes.     

Salt induced B----A transition of poly(dG).poly(dC) and the stabilization of A form by its methylation.

Raman spectra of poly(dG).poly(dC) have been observed in aqueous solutions at various ionic strengths, [NaCl] = 0.03 to 4 M, and at different temperatures, 10 to 60 degrees C. At 30 degrees C, and at [NaCl] = 0.03 M, it was found to have a B-form (with O4'endo-anti guanosine and C2'endo-anti cytidine), whereas, at [NaCl] = 4 M, an A form (with C3'endo-anti guanosine and C3'endo-anti cytidine). At 30 degrees C and [NaCl] = 1 M, namely at an intermediate state, a fraction of this molecules was considered to have a "heteronomous A" form (with O4'endo-anti guanosine and C3' endo-anti cytidine). At 60 degrees C and [NaCl] = 1 M, it assumes the B form, and at 10 degrees C and [NaCl] = 1 M, the A form. Cytosine-5-methylation was found to cause a marked stabilization of the A form. Even at [NaCl] = 0.1 M (at 30 degrees C), a substantial portion of poly(dG).poly(dm5C) was found to have a heteronomous form, in which the dG atrand is in the B form and the dC an A form; it never assumes a complete B form.     

Nucleosome reconstitution of core-length poly(dG).poly(dC) and poly(rG-dC).poly(rG-dC)

The double-stranded polypurine.polypyrimidines poly(dG).poly(dC) and poly[d(A-G)].poly[d(T-C)] and the mixed ribose-deoxyribose polynucleotide poly(rG-dC).poly(rG-dC) have been successfully reconstituted into nucleosomes. The radioactively labeled particles comigrate in gel electrophoresis and sucrose density gradient experiments with authentic nucleosomes derived from chicken erythrocyte chromatin. These results show that nucleosomes are able to accommodate a wider variety of polynucleotides than was previously believed.     

The molecular electrostatic potential and steric accessibility of poly (dI.dC). Comparison with poly (dG.dC)

The influence of the amino group of guanine on the molecular electrostatic potential and the accessibility to reactive sites of B-DNA is investigated by comparing the two model double helices poly (dI.dC) and poly (dG.dC). The calculations clarify the “disruptive” role of the guanine amino group on nucleic acid-polypeptide interactions.     

A parallel stranded linear DNA duplex incorporating dG.dC base pairs

DNA oligonucleotides with appropriately designed complementary sequences can form a duplex in which the two strands are paired in a parallel orientation and not in the conventional antiparallel double helix of B-DNA. All parallel stranded (ps) molecules reported to date have consisted exclusively of dA.dT base pairs. We have substituted four dA.dT base pairs of a 25-nt parallel stranded linear duplex (ps-D1.D2) with dG.dC base pairs. The two strands still adopt a duplex structure with the characteristic spectroscopic properties of the ps conformation but with a reduced thermodynamic stability. Thus, the melting temperature of the ps duplex with four dG.dC base pairs (ps-D5.D6) is 10-16 degrees C lower and the van't Hoff enthalpy difference delta HvH for the helix-coil transition is reduced by 20% (in NaCl) and 10% (in MgCl2) compared to that of ps-D1.D2. Based on energy minimizations of a ps-[d(T5GA5).d(A5CT5)] duplex using force field calculations we propose a model for the conformation of a trans dG.dC base pair in a ps helix.     

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.     

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.     

Crystallization of immunoglobulin Fab fragments specific for DNA

The purification and crystallization of Fab fragments of two mouse monoclonal immunoglobulins specific for different DNA structures are described. In each case, papain digestion of the immunoglobulins produced a mixture of Fab species differing in their isoelectric points. Purification of one of these species was required to obtain suitable crystals. One of these antibodies, Jel 72, is specific for right-handed duplex poly(dG).poly(dC). An Fab fragment of Jel 72 with a pI of 8.8 was purified by anion-exchange chromatography and used to obtain crystals from 56% saturated ammonium sulfate and 50 mM sodium acetate, pH 4.2, that diffract to 2.6-A resolution. They belong to the orthorhombic space group P2(1)2(1)2(1), with cell dimensions of a = 94.6, b = 102.6, c = 92.4 A. The other antibody, Jel 318, binds triple-stranded DNA poly[d(Tm5C)].poly[d(GA)].poly[d(m5C + T)]. Jel 318 Fab fragments with isoelectric points of 7.6 and 7.8 were also purified by anion-exchange chromatography, and crystals were obtained from 12% polyethylene glycol 8000, 50 mM NaCl, and 10 mM Tris.HCl, pH 7.8. These crystals diffract to about 2.4-A resolution and also belong to the orthorhombic space group P2(1)2(1)2(1), with cell dimensions of a = 82.4, b = 139.5, and c = 42.0 A. For both Fab fragments, crystal size and quality improved dramatically upon purification of an individual isoelectric species.     

Poly(dG).poly(dC) at neutral and alkaline pH: the formation of triple stranded poly(dG).poly(dG).poly(dC).

Alkaline titrations of different samples of poly(dG).poly(dC) and of the constituent homopolymers poly(dG) and poly(dC) have been performed in 0.15 M NaCl and their CD spectra followed. Sample I contained a slight excess of poly(dC) (52% C: 48% G) and showed a single reversible transition (pK = 11.9) due to the dissociation of double stranded poly(dG).poly(dC). Sample II, containing an excess of poly(dG) (43% C: 57% G), showed two transitions (pK1 = 11.4, PK2 = 11.9) the first one being only partially reversible. Examination of the CD spectra along the alkaline titrations indicated the presence of another hydrogen-bonded complex of higher G content. Mixing curves performed at pH 8 have confirmed the presence of a 2G: 1C complex, besides the double stranded complex. It can be formed in amounts up to 30% by mixing the two homopolymers, alkali treatment and heating. The CD spectra of the two complexes have been computed from the CD data of the mixing curves. This permitted the determination of the concentrations of both complexes and homopolymers in all samples. The ratio of triple to double stranded complex is not only dependent on the G/C ratio of the sample, but also a function of the previous physico-chemical conditions. These results explain the variability of many properties of different poly(dG).poly(dC) samples observed by other workers.     

Binding mode of 4',6-diamidino-2-phenylindole and ethidium to poly(dG).poly(dC).poly(dC)(+) triplex and poly(dG).poly(dC) duplex

Optical spectroscopic properties of 4',6-diamidino-2-phenylindole (DAPI) and ethidium bromide complexed with poly(dG).poly(dC).poly(dC)(+) triplex and poly(dG).poly(dC) duplex were compared in this study. When complexed with both duplex and triplex, ethidium is characterized by hypochromism and a red shift in the absorption spectrum, a complicate induced circular dichroism (CD) band in the polynucleotide absorption region, and a negative reduced linear dichroism signal in both polynucleotide and drug absorption regions. The spectral properties for both duplex- and triplex-bound ethidium are identical and both can be understood by the intercalation binding mode. In contrast, the absorption and CD spectra of DAPI complexed with triplex differ from those of the DAPI-duplex complex, although both complexes can be understood by the intercalation binding mode. Considering that the third strand runs along the major groove of the template duplex, we conclude that the DAPI molecule partially intercalates near the major groove of the duplex, where the third strand can affect its spectroscopic properties.