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.     

NMR studies of the exocyclic 1,N6-ethenodeoxyadenosine adduct (epsilon dA) opposite deoxyguanosine in a DNA duplex. Epsilon dA(syn).dG(anti) pairing at the lesion site

Proton NMR studies are reported on the complementary d(C-A-T-G-G-G-T-A-C).d(G-T-A-C-epsilon A-C-A-T-G) nonanucleotide duplex (designated epsilon dA.dG 9-mer duplex), which contains exocyclic adduct 1,N6-ethenodeoxyadenosine positioned opposite deoxyguanosine in the center of the helix. The present study focuses on the alignment of dG5 and epsilon dA14 at the lesion site in the epsilon dA.dG 9-mer duplex at neutral pH. This alignment has been characterized by monitoring the NOEs originating from the NH1 proton of dG5 and the H2, H5, and H7/H8 protons of epsilon dA14 in the central d(G4-G5-G6).d(C13-epsilon A14-C15) trinucleotide segment of the epsilon dA.dG 9-mer duplex. These NOE patterns establish that epsilon dA14 adopts a syn glycosidic torsion angle that positions the exocyclic ring toward the major groove edge while all the other bases including dG5 adopt anti glycosidic torsion angles. We detect a set of intra- and interstrand NOEs between protons (exchangeable and nonexchangeable) on adjacent residues in the d(G4-G5-G6).d(C13-epsilon A14-C15) trinucleotide segment which establish formation of right-handed helical conformations on both strands and stacking of the dG5(anti).epsilon dA14(syn) pair between stable dG4.dC15 and dG6.dC13 pairs. The energy-minimized conformation of the central d(G4-G5-G6).d(C13-epsilon A14-C15) segment establishes that the dG5(anti).epsilon dA14(syn) alignment is stabilized by two hydrogen bonds from the NH1 and NH2-2 of dG5(anti) to N9 and N1 of epsilon dA14(syn), respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

NMR studies of the exocyclic 1,N6-ethenodeoxyadenosine adduct (epsilon dA) opposite thymidine in a DNA duplex. Nonplanar alignment of epsilon dA(anti) and dT(anti) at the lesion site

Two-dimensional proton NMR studies are reported on the complementary d(C-A-T-G-T-G-T-A-C).d(G-T-A-C-epsilon A-C-A-T-G) nonanucleotide duplex (designated epsilon dA.dT 9-mer duplex) containing 1,N6-ethenodeoxyadenosine (epsilon dA), a carcinogen-DNA adduct, positioned opposite thymidine in the center of the helix. Our NMR studies have focused on the conformation of the epsilon dA.dT 9-mer duplex at neutral pH with emphasis on defining the alignment at the dT5.epsilon dA14 lesion site. The through-space NOE distance connectivities establish that both dT5 and epsilon dA14 adopt anti glycosidic torsion angles, are directed into the interior of the helix, and stack with flanking Watson-Crick dG4.dC15 and dG6.dC13 pairs. Furthermore, the d(G4-T5-G6).d(C13-epsilon A14-C15) trinucleotide segment centered about the dT5.epsilon dA14 lesion site adopts a right-handed helical conformation in solution. Energy minimization computations were undertaken starting from six different alignments of dT5(anti) and epsilon dA14(anti) at the lesion site and were guided by distance constraints defined by lower and upper bounds estimated from NOESY data sets on the epsilon dA.dT 9-mer duplex. Two families of energy-minimized structures were identified with the dT5 displaced toward either the flanking dG4.dC15 or the dG6.dC13 base pair. These structures can be differentiated on the basis of the observed NOEs from the imino proton of dT5 to the imino proton of dG4 but not dG6 and to the amino protons of dC15 but not dC13 that were not included in the constraints data set used in energy minimization. Our NMR data are consistent with a nonplanar alignment of epsilon dA14(anti) and dT5(anti) with dT5 displaced toward the flanking dG4.dC15 base pair within the d(G4-T5-G6).d(C13-epsilon A14-C15) segment of the epsilon dA.dT 9-mer duplex.     

Conformational properties of poly[d(G-T)].poly[d(C-A)] and poly[d(A-T)] in low- and high-salt solutions: NMR and laser Raman analysis

³¹P- and ¹H-nmr and laser Raman spectra have been obtained for poly[d(G-T)]·[d(C-A)] and poly[d(A-T)] as a function of both temperature and salt. The ³¹P spectrum of poly[d(G-T)]·[d(C-A)] appears as a quadruplet whose resonances undergo separation upon addition of CsCl to 5.5M. ¹H-nmr measurements are assigned and reported as a function of temperature and CsCl concentration. One dimensional nuclear Overhauser effect (NOE) difference spectra are also reported for poly[d(G-T)]·[d(C-A)] at low salt. NOE enhancements between the H8 protons of the purines and the C5 protons of the pyrimidines, (H and CH₃) and between the base and H-2′,2″ protons indicate a right-handed B-DNA conformation for this polymer. The NOE patterns for the TH3 and GH1 protons in H₂O indicate a Watson–Crick hydrogen-bonding scheme. At high CsCl concentrations there are upfield shifts for selected sugar protons and the AH2 proton. In addition, laser Raman spectra for poly[d(A-T)] and poly[d(G-T)]·[d(C-A)] indicate B-type conformations in low and high CsCl, with predominantly C2′-endo sugar conformations for both polymers. Also, changes in base-ring vibrations indicate that Cs⁺ binds to O2 of thymine and possibly N3 of adenine in poly[d(G-T)]·[d(C-A)] but not in poly[d(A-T)]. Further, ¹H measurements are reported for poly[d(A-T)] as a function of temperature in high CsCl concentrations. On going to high CsCl there are selective upfield shifts, with the most dramatic being observed for TH1′. At high temperature some of the protons undergo severe changes in linewidths. Those protons that undergo the largest upfield shifts also undergo the most dramatic changes in linewidths. In particular TH1′, TCH₃, AH1′, AH2, and TH6 all undergo large changes in linewidths, whereas AH8 and all the H-2′,2″ protons remain essentially constant. The maximum linewidth occurs at the same temperature for all protons (65°C). This transition does not occur for d(G-T)·d(C-A) at 65°C or at any other temperature studied. These changes are cooperative in nature and can be rationalized as a temperature-induced equilibrium between bound and unbound Cs⁺, with duplex and single-stranded DNA. NOE measurements for poly[d(A-T)] indicate that at high Cs⁺ the polymer is in a right-handed B-conformation. Assignments and NOE effects for the low-salt ¹H spectra of poly[d(A-T)] agree with those of Assa-Munt and Kearns [(1984) Biochemistry 23 , 791–796] and provide a basis for analysis of the high Cs⁺ spectra. These results indicate that both polymers adopt a B-type conformation in both low and high salt. However, a significant variation is the ability of the phosphate backbone to adopt a repeat dependent upon the base sequence. This feature is common to poly[d(G-T)]·[d(C-A)], poly[d(A-T)], and some other pyr–pur polymers [J. S. Cohen, J. B. Wouten & C. L Chatterjee (1981) Biochemistry 20 , 3049–3055] but not poly[d(G-C)].     

DNA fragment conformations. A 1H-NMR conformational analysis of the d(G-G)-chelated platinum-oligonucleotide d(A-T-G-G)cisPt

The conformation of d(A-T-G-G) and d(A-T-G-G)cisPt has been investigated by 1H-NMR at 500 MHz and 90 MHz under various experimental conditions of temperature and concentration. Analysis of the coupling constants between the deoxyribose protons shows that all the sugar rings of d(A-T-G-G) adopt the S(C2'-endo) conformation most of the time. By contrast, in the platinated tetramer, d(A-T-G-G)cisPt, the N(C3'-endo) conformation is highly predominant for the internal dG residue while the S(C2'-endo) conformation is largely favoured for the other residues as in the case of the unplatinated compound. The relaxation time and nuclear Overhauser effect measurements indicate that the orientation of the two guanines of d(A-T-G-G)cisPt is anti in agreement with the previous results obtained for the dimers: r(G-G)cisPt, d(G-G)cisPt. On lowering the temperature from 80 degrees C to 20 degrees C, several proton resonances of d(A-T-G-G)cisPt exhibit large chemical shift and linewidth variations. The most spectacular temperature effect was observed for the internal dG(H1') and dT(H4') protons. All the delta = f(t) curves display a sigmoid form with the same mid-point temperature of 44 +/- 2 degrees C. This mid-point temperature together with the observed chemical shift and linewidth variations were found to be independent of the d(A-T-G-G)cisPt concentration. These results suggest that d(A-T-G-G)cisPt can adopt two different conformations depending on the temperature. The enthalpy for the transition between the high and low temperature conformations is about 84 kJ/mol.     

Z-DNA conformation of N-2-acetylaminofluorene modified poly(dG-dC).poly(dG-dC) determined by reactivity with anti cytidine antibodies and minimized potential energy calculations.

The conformation of poly(dG-dC).poly(dG-dC), poly(dG).poly(dC), and calf thymus DNA modified with N-acetoxy-N-2-acetylaminofluorene (N-acetoxy-AAF) was examined by extent of reaction with anti cytidine antibodies. In contrast to modified poly(dG).poly(dC0 and DNA, modified poly(dG-dC).poly (dG-dC) failed to react with the antibodies indicating that the base pairing in this polymer is intact. This in consistent with induction of the Z-DNA conformation in AAF modified poly(dG-dC).poly(dG-dC). Using minimized potential energy calculations on the dCpdG-AAF dimer as a model for the modified polymer, it is shown that the proposed Z-DNA conformation is energetically stable. A model is proposed for an AAF modified tetramer, dGpdCpdGpdC, in which the AAF is external to the Z-DNA duplex.     

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.     

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.     

Deoxyguanosine-deoxyadenosine pairing in the d(C-G-A-G-A-A-T-T-C-G-C-G) duplex: conformation and dynamics at and adjacent to the dG X dA mismatch site

Nuclear magnetic resonance (NMR) has been used to monitor the conformation and dynamics of the d-(C1-G2-A3-G4-A5-A6-T6-T5-C4-G3-C2-G1) self-complementary dodecanucleotide (henceforth called 12-mer GA) that contains a dG X dA purine-purine mismatch at position 3 in the sequence. These results are compared with the corresponding d(C-G-C-G-A-A-T-T-C-G-C-G) dodecamer duplex (henceforth called 12-mer) containing standard Watson-Crick base pairs at position 3 [Patel, D.J., Kozlowski, S.A., Marky, L.A., Broka, C., Rice, J.A., Itakura, K., & Breslauer, K.J. (1982) Biochemistry 21, 428-436]. The dG X dA interaction at position 3 was monitored at the guanosine exchangeable H-1 and nonexchangeable H-8 protons and the nonexchangeable adenosine H-2 proton. We demonstrate base-pair formation between anti orientations of the guanosine and adenosine rings on the basis of nuclear Overhauser effects (NOE) observed between the H-2 proton of adenosine 3 and the imino protons of guanosine 3 (intra base pair) and guanosines 2 and 4 (inter base pair). The dG(anti) X dA(anti) pairing should result in hydrogen-bond formation between the guanosine imino H-1 and carbonyl O-6 groups and the adenosine N-1 and NH2-6 groups, respectively. The base pairing on either side of the dG X dA pair remains intact at low temperature, but these dG X dC pairs at positions 2 and 4 are kinetically destabilized in the 12-mer GA compared to the 12-mer duplex. We have estimated the hydrogen exchange kinetics at positions 4-6 from saturation-recovery measurements on the imino protons of the 12-mer GA duplex between 5 and 40 degrees C. The measured activation energies for imino proton exchange in the 12-mer GA are larger by a factor of approximately 2 compared to the corresponding values in the 12-mer duplex. This implies that hydrogen exchange in the 12-mer GA duplex results from a cooperative transition involving exchange of several base pairs as was previously reported for the 12-mer containing a G X T wobble pair at position 3 [Pardi, A., Morden, K.M., Patel, D.J., & Tinoco, I., Jr. (1982) Biochemistry 21, 6567-6574]. We have assigned the nonexchangeable base protons by intra and inter base pair NOE experiments and monitored these assigned markers through the 12-mer GA duplex to strand transition.(ABSTRACT TRUNCATED AT 400 WORDS)     

1H two-dimensional nuclear Overhauser effect and relaxation studies of poly(dA).poly(dT)

The structure of poly(dA).poly(dT) in aqueous solution has been studied by using 1H two-dimensional nuclear Overhauser effect (2D NOE) spectroscopy and relaxation rate measurements on the imino and nonexchangeable protons. The assignments of the 1H resonances are determined from the observed cross-relaxation patterns in the 2D NOE experiments. The cross-peak intensities together with the measured relaxation rates show that the purine and pyrimidine strands in poly(dA).poly(dT) are equivalent in aqueous solution. The results are consistent with a right-handed B-form helix where the sugars on both strands are in the C2'-endo/anti configuration. These observations are inconsistent with a proposed heteronomous structure for poly(dA).poly(dT) [Arnott, S., Chandrasekaran, R., Hall, I. H., & Puigjaner, L. C. (1983) Nucleic Acids Res. 11, 4141-4155]. The measured relaxation rates also show that poly(dA).poly(dT) has fast, large-amplitude local internal motions (+/- 20-25 degrees) in solution and that the amplitudes of the base and sugar motions are similar. The motion of the bases in poly(dA).poly(dT) is also similar to that previously reported for poly(dA-dT).poly(dA-dT) and poly(dG-dC).poly(dG-dC) [Assa-Munt, N., Granot, J., Behling, R. W., & Kearns, D. R. (1984) Biochemistry 23, 944-955; Mirau, P. A., Behling, R. W., & Kearns, D. R. (1985) Biochemistry 24, 6200-6211].     

Conversions of poly(2-aminodeoxyadenylate-5-halodeoxyuridylate) from B to A forms in high salt. An NMR and circular dichroism study

Proton one- and two-dimensional nuclear Overhauser enhancement (1D and 2D NOE) spectroscopy has been used to demonstrate that poly(d2NH2A-d5IU) and poly(d2NH2A-d5BrU) are converted from the B to the A conformation in high salt, as found previously for poly(d2NH2A-dT) [Borah, B., Cohen, J. S., Howard, F. B., & Miles, H. T. (1985) Biochemistry 24, 7456-7462]. The 2D NOE and 1D NOE spectra exhibit strong base proton (H8,H6)-H3' cross relaxation, suggesting short interproton distances. These results are indicative of a C3'-endo sugar pucker for both purine and pyrimidine residues in an A or closely related structure. The circular dichroism and UV spectra are consistent with the interpretation of an A conformation in high salt.     

DNA hairpin structures in solution: 500-MHz two-dimensional 1H NMR studies on d(CGCCGCAGC) and d(CGCCGTAGC)

A hairpin structure contains two conformationally distinct domains: a double-helical stem with Watson-Crick base pairs and a single-stranded loop that connects the two arms of the stem. By extensive 1D and 2D 500-MHz 1H NMR studies in H2O and D2O, it has been demonstrated that the DNA oligomers d(CGCCGCAGC) and d(CGCCGTAGC) form hairpin structures under conditions of low concentration, 0.5 mM in DNA strand, and low salt (20 mM NaCl, pH 7). From examination of the nuclear Overhauser effect (NOE) between base protons H8/H6 and sugar protons H1' and H2'/H2", it was concluded that in d(CGCCGCAGC) and d(CGCCGTAGC) all the nine nucleotides display average (C2'-endo,anti) geometry. The NMR data in conjunction with molecular model building and solvent accessibility studies were used to derive a working model for the hairpins.     

Assignment of non-exchangeable base proton and H1' resonances of a deoxyoctanucleoside heptaphosphate d(G-G-C*-C*-G-G-C-C) by using the nuclear Overhauser effect.

The resonances of the non-exchangeable base protons and 1' protons of the octamer d(G-G-C*-C*-G-G-C-C), C* = m5dC, have been assigned by means of NOE difference NMR spectroscopy at 500 MHz. From the measured J1'2' and J1'2" it follows that the octamer at low temperature prefers to adopt a B-DNA double-helical conformation in solution, however, some residual conformational freedom is detected at the 3' terminus. From the chemical shift versus temperature profiles it is concluded that no major conformational change occurs below 60-65 degrees C where the duplex formation for residues (2) to (6) is essentially completed under the conditions used.     

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.     

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.     

Sequence-dependent recognition of DNA duplexes. Netropsin complexation to the AATT site of the d(G-G-A-A-T-T-C-C) duplex in aqueous solution

We have investigated intermolecular interactions and conformational features of the netropsin X d(G-G-A-A-T-T-C-C) complex by one- and two-dimensional NMR studies in aqueous solution. Netropsin removes the 2-fold symmetry of the d(G-G-A-A-T-T-C-C) duplex at the AATT binding site and to a lesser extent at adjacent dG X dC base pairs resulting in doubling of resonances for specific positions in the spectrum of the complex at 25 degrees C. We have assigned the amide, pyrrole, and CH2 protons of netropsin, and the base and sugar H1' protons of the nucleic acid from an analysis of the nuclear Overhauser effect (NOESY) and correlated (COSY) spectra of the complex at 25 degrees C. We observe intermolecular nuclear Overhauser effects (NOE) between all three amide and both pyrrole protons on the concave face of the antibiotic and the minor groove adenosine H2 proton of the two central A4 X T5 base pairs of the d(G1-G2-A3-A4-T5-T6-C7-C8) duplex. Weaker intermolecular NOEs are also observed between the pyrrole concave face protons and the sugar H1' protons of residues T5 and T6 in the AATT minor groove of the duplex. We also detect intermolecular NOEs between the guanidino CH2 protons at one end of netropsin and adenosine H2 proton of the two flanking A3 X T6 base pairs of the octanucleotide duplex. These studies establish a set of intermolecular contacts between the concave face of the antibiotic and the minor groove AATT segment of the d(G-G-A-A-T-T-C-C) duplex in solution. The magnitude of the NOEs require that there be no intervening water molecules sandwiched between the antibiotic and the DNA so that release of the minor groove spine of hydration is a prerequisite for netropsin complex formation.     

Secondary structure of the hybrid poly(rA).poly(dT) in solution. Studies involving NOE at 500 MHz and stereochemical modelling within the constraints of NOE data

One-dimensional nuclear Overhauser effect (NOE) in nuclear magnetic resonance spectroscopy along with stereochemically sound model building was employed to derive the structure of the hybrid poly(rA).poly(dT) in solution. Extremely strong NOE was observed at AH2' when AH8 was presaturated; strong NOEs were observed at TH2'TH2' when TH6 was presaturated; in addition the observed NOEs at TH2' and TH2' were nearly equal when TH6 was presaturated. There was no NOE transfer to AH3' from AH8 ruling out the possibility of (C-3'-endo, low anti chi approximately equal to 200 degrees to 220 degrees) conformation for the A residues. The observed NOE data suggest that the nucleotidyl units in both rA and dT strands have equivalent conformations: C-2'-endo/C-1'-exo, anti chi approximately equal to 240 degrees to 260 degrees. Such a nucleotide geometry for rA/dT is consistent with a right-handed B-DNA model for poly(rA).poly(dT) in solution in which the rA and dT strands are conformationally equivalent. Molecular models were generated for poly(rA).poly(dT) in the B-form based upon the geometrical constraints as obtained from the NOE data. Incorporation of (C-2'-endo pucker, chi congruent to 240 degrees to 260 degrees) into the classical B-form resulted in severe close contacts in the rA chain. By introducing base-displacement, tilt and twist along with concomitant changes in the backbone torsion angles, we were able to generate a B-form for the hybrid poly(rA).poly(dT) fully consistent with the observed NOE data. In the derived model the sugar pucker is C-1'-exo, a minor variant of C-2'-endo and the sugar base torsion is 243 degrees, the remaining torsion angles being: epsilon = 198 degrees, xi = 260 degrees, alpha = 286 degrees, beta = 161 degrees and gamma = 72 degrees; this structure is free of any steric compression and indicates that it is not necessary to switch to C-3'-endo pucker for rA residues in order to accommodate the 2'-OH group. The structure that we have proposed for the polynucleotide RNA-DNA hybrid in solution is in complete agreement with that proposed for a hexamer hybrid in solution from NOE data and is inconsistent with the heteronomous model proposed for the fibrous state.     

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.     

Conformation of guanosine 5'-diphosphate as bound to a human c-Ha-ras mutant protein: a nuclear Overhauser effect study

1H NMR spectra of a GDP/GTP-binding domain of human c-Ha-ras gene product (residues 1-171) in which glutamine-61 was replaced by leucine [ras(L61/1-171) protein] were analyzed. By one-dimensional and two-dimensional homonuclear Hartmann-Hahn spectroscopy and nuclear Overhauser effect (NOE) spectroscopy of the complex of the ras(L61/1-171) protein and GDP, the ribose H1', H2', H3', and H4' proton resonances of the bound GDP were identified. The guanine H8 proton resonance of the bound GDP was identified by substituting [8-2H]GDP for GDP. The dependences of the H1' and H8 proton resonance intensities on the duration of irradiation of the H1', H2', H3', and H8 protons were measured. By numerical simulation of these time-dependent NOE profiles, the conformation of the protein-bound GDP was elucidated; the guanosine moiety takes the anti form about the N-glycosidic bond with a dihedral angle of chi = -124 +/- 2 degrees and the ribose ring takes the C2'-endo form. Such an analysis of the conformation of a guanine nucleotide as bound to a GTP-binding protein will be useful for further studies on the molecular mechanism of the conformational activation of ras proteins on ligand substitution of GDP with GTP.     

The complex of poly(dG).poly(dC) with arginine: stabilization of the B form and transition to multistranded structures

We have studied by X-ray diffraction fibers of complexes of poly(dG).poly(dC) with N-alpha-acetyl-L-arginine ethylamide. Although these polynucleotides favour the A form of DNA, in this complex it is never found, thus confirming that arginine prevents the appearance of this form of DNA. At high relative humidity the B form is present. Upon dehydration two new structures appear. One of them is a triple helix, most likely formed by poly(dC+).poly(dG).poly(dC). The other structure found also has features which indicate a multistranded conformation.