Effect of 5-alkyl substitution of uracil on the thermal stability of poly [d(A-r5U)] copolymers.

The thermal transition of poly[d(A-r5U)] polydeoxynucleotides (where r was a hydrogen atom, or a methyl, ethyl, n-propyl, n-butyl or n-pentyl group) was studied by measuring the derivative melting profiles of the polymers in the range of 0.01--0.36 M K+, at pH 6.8. According to the Tm values, polydeoxynucleotide analogues show lower thermal stability than poly[d(A-T)] at any counterion concentration applied. At a given salt concentration, Tm of the alkyl analogues decreased as the number of carbon atoms (n) in the r substituent of poly[d(A-r5U)] increased. 1/Tm plotted against against 1/n yielded a linear relationship. Cooperativity of the melting of all poly[d(A--U)] analogues decreased with the increase of salt concentration in the solution. This change depended again on 5-substitution of the uracil moiety of poly[d(A-U)]. Smallest decrease was observed in the case of poly[d(A--U)] whereas largest decrease was shown by poly[d(A-pe5U)] (pe=pentyl group).     

Modified polynucleotides. V. Slow-down of nuclease action by 5-alkyluracil-containing DNAs

Poly d/[³H]A-r⁵U/ type of synthetic models of bacteriophage DNAs containing thymine analogues were prepared by DNA polymerase and tested for stability against nucleases /$\text{r}$ was a n-alkyl group from methyl to pentyl/. The 5-pentyluracil-containing copolymer was found to be most stable: 50 % degradation with pancreatic DNase, spleen DNase, snake venom phosphodiesterase or micrococcal nuclease required 3–15 times as much time as that of poly d/A-T/.     

Proflavin binding to poly[d(A-T)] and poly[d(A-br5U)]: triplet state and temperature-jump kinetics

The delayed fluorescence properties of proflavin have been exploited in studies of the excited-state binding kinetics of the dye to poly[d(A-T)] and its brominated analogue poly[d(A-br5U)] at room temperature and pH 7. The two analyzed luminescence decay times of the DNA-dye complex are dependent on the total nucleic acid concentration. This dependence is shown to reflect a temporal coupling of the intrinsic delayed emission decay rates with the dynamic chemical kinetic binding processes in the excited state. Temperature-jump kinetic studies conducted on the brominated polymer and corresponding information on poly[d(A-T)] from a previous study [Ramstein, J., Ehrenberg, M., & Rigler, R. (1980) Biochemistry 19, 3938-3948] provide complementary information about the ground state. In the ground state, the poly[d(A-T)]-proflavin complex has one chemical relaxation time, which reaches a plateau at high DNA concentrations. The brominated DNA-dye complex exhibits two relaxation times: a faster relaxation mode that behaves similarly to that for the unhalogenated DNA and a slower relaxation mode that is apparent at high DNA concentrations. The ground-state kinetic data are analyzed in terms of two alternative models incorporating series and parallel reaction schemes. The former consists of two sequential binding steps--a fast bimolecular process followed by a monomolecular step--while the latter consists of two coupled bimolecular steps. A similar analysis for the excited-state data yields reasonable kinetic constants only for the series model, which, in accordance with previous proposals for acridine intercalators, consists of a fast outside binding step followed by intercalation of the dye. A comparison of the ground- and excited-state kinetic parameters reveals that the external binding process is much stronger and the intercalation is much weaker in the excited state. That the excited-state data are only consistent with the series model suggests that delayed luminescence studies may provide a general tool for distinguishing between the two kinetic mechanisms. In particular, we demonstrate the use of delayed luminescence spectroscopy as a tool for probing dynamic DNA-ligand interactions in solution.     

Nitrogen mustard fixes the Z-conformation in poly[d(G-C)]poly[d(G-C)] and DNA]

The reactions of poly(dG-dC).poly(dG-dC) and (dG-dC)10 insert in the plasmid pGC20 with N-methyl-bis(2-chloroethyl)-amine (nitrogen mustard, HN-2) have been studied. It is shown that nitrogen mustard does not induce the B----Z transition in poly(dG-dC).poly(dG-dC), but produces fixation of the polynucleotide Z-conformation once this exists. In the case of pGC20 plasmid DNA, nitrogen mustard also fixes Z-form of the (dG-dC)-insert. The rate constant of the reaction of nitrogen mustard with guanine in the polynucleotide (k = 9,0.10(-3) min-1) is about one-third of that for the fixation of Z-form of the (dG-dC)-insert in the plasmid (k1 = 2,8.10(-2) min-1) which is attributed to a greater rate of formation of diguanyl derivative in the opposite DNA chains. It is suggested that nitrogen mustard is capable of fixing the Z-form DNA not only in vitro, but also in vivo.     

Infrared dichroism of polynucleotides of repeated sequence. I. Poly(dA)·poly(dT) and poly[d(A-T)]·poly[d(A-T)]

Infrared dichroism measurements of oriented films of poly(dA)·poly(dT) and poly[d(A-T)]·poly[d(A-T)] have been made under the conditions of low salts content and high humidity for which the geometry is known. The angles which the transition moments make with the helix axis are compared with the orientations of the corresponding bonds. Except for the in-plane base model of poly[(A-T)]·poly[d(A-T)], there is no agreement. This may imply either that a model which assumes bonds and transition moments to be colinear is not acceptable or that x-ray data are inaccurate. These possibilities are discussed especially with respect to phosphate group orientation. An appendix gives the derivations of dichroic-ratio expressions for helical molecules of different symmetry types.     

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.     

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.     

Base tilt of poly[d(A)]-poly[d(T)] and poly[d(AT)]-poly[d(AT)] in solution determined by linear dichroism

We have measured the CD, isotropic absorption, and LD of poly[d(A)]–poly[d(T)] and poly[d(AT)]–poly[d(AT)] in the vacuum-uv spectral region. The reduced dichroism (LD divided by isotropic absorption) varied as a function of wavelength and was independent of shear gradient. Thus, the bases are not perpendicular to the helix axis in solution. Since the directions of the transition dipoles are known, the orientations of the bases in the polymers can be calculated from the reduced dichroism spectra. The results show that the base normals are tilted at angles greater than 25°, with respect to the helix axis, and thymine is tilted more than adenine for both polymers. The tilt axes of adenine and thymine are not parallel, indicating a large propeller twist. Space-filling models of poly[d(A)]–poly[d(T)] and poly[(AT)]–poly[d(AT)] are built based on our results, and the conformations of the two (A + T) polymers in solution are discussed.     

Base tilt of B-form poly[d(G)]-poly[d(C)] and the B- and Z-conformations of poly[d(GC)]-poly[d(GC)] in solution

We have measured the CD, isotropic absorption, and linear dichroism (LD) in the vacuum-uv spectral region for the B-conformations of poly[d(G)]-poly[d(C)] and poly[d(GC)]-poly[d(GC)], and for the Z-conformation of poly[d(GC)]-poly[d(GC)] formed in 70% trifluoroethanol. The reduced dichroism (LD divided by isotropic absorption) for all conformations varied with wavelength, indicating that the bases are not perpendicular to the helix axis. Since the directions of the transition dipoles are known, the inclinations and axes of inclination of each base can be determined from the wavelength dependence of the reduced dichroism spectra. The results indicate that the base normals of the (G + C) polymers in the B- and Z-conformations are tilted at angles greater than 19° with respect to the helix axis. The guanine and cytosine bases have different inclinations, and the tilt axes are not parallel. Therefore, the bases for all the (G + C) polymer conformations studied are buckled and propeller twisted.     

Elsamicin A can convert the Z-form of poly[d(G-C)] and poly[(G-m5C)] back to B-form DNA.

The interaction of poly[(G-C)] and poly[d(G-m5C)] with the antitumor antibiotic elsamicin A, which binds to alternating guanine + cytosine tracts in DNA, has been studied under the B and Z conformations. Both the rate and the extent of the B-to-Z transition are diminished by the antibiotic, as inferred by spectroscopic methods under ionic conditions that otherwise favor the left-handed conformation of the polynucleotides. Moreover, elsamicin converts the Z-form DNA back to the B-form. The circular dichroism data indicate that elsamicin binds to poly[d(G-C)] and poly[d(G-m5C)] to form a right-handed bound elsamicin region(s). The transition can be followed by changes of the molar ellipticity at 250 nm, thus providing a convenient wavelength to monitor the Z-to-B conformational change of the polymers as elsamicin is added. The elsamicin A effect might be explained by a model in which the antibiotic binds preferently to a B-form DNA, playing a role as an allosteric effector on the equilibrium between the B and Z conformations, thus favoring the right-handed one.     

Psi compaction of poly[d(AT)].poly[d(AT)]

The compaction of poly[d(A–T)] · poly[d(A–T)] by Co(III) is accompanied by the formation of ψ(+)- and ψ(-)-structures. The chirality of the ψ-structure depends on the Co(III) concentration, ionic strength, temperature, pH, and the chain length of the polymer. The two forms can be readily interconverted by manipulating these factors. Phase diagrams have been constructed that demonstrate the regions of stability of the enantiomers as a function of two variables, while other factors are held constant. At critical points in the phase diagram the two forms are in such unstable equilibrium that mechanical motion will cause ψ(+) ? ψ(-) interconversion. The formation of both ψ(+)- and ψ(-)-structures by the action of Co(III) on poly[d(A–T)] · poly[d(A–T)] contrasts markedly with the behavior of poly[d(G–C)] · poly[d(G–C)] in similar circumstances by forming only the ψ(+)-structure and that of native DNA to produce no ψ at all. Thus the base sequence is important in determining the structure of chirally associated DNA molecules.     

The synthetic DNA duplex of poly d(Abr5U).poly d(Abr5U) adopts an A-DNA-like structure

An X-ray fiber diffraction study of the synthetic DNA duplex poly d(Abr5U).poly d(Abr5U) shows that its sodium salt adopts an unexceptional A-DNA-like structure. Similar to A-DNA, two molecules are packed in a monoclinic unit cell (a = 2.23 nm, b = 4.14 nm, c = 5.61 nm and alpha = beta = gamma = 90 degrees) of space group C2. Because of its dinucleotide chemical motif, the c-repeat is twice that in A-DNA but, notably, corresponding backbone conformation angles of adjacent nucleotides are almost identical. This is in marked contrast to many B-like conformations of polydinucleotides.     

C4-methyldeoxythymidine replacing deoxythymidine in poly[d(A-T)] renders the polymer resistant to the 3''----5'' exonuclease activity of the Klenow and T4 DNA polymerases.

We previously reported that O4-alkyl dTTPs could replace, for short times, dTTP in polymer synthesis [Singer et al., PNAS 83, 26-32, 1986]. The reasons for such early termination of synthesis could be either proofreading or the eventual formation of weakly paired primer termini. Utilizing the known 3'----5' exonucleolytic activity of polymerases, in the absence of dNTPs, enabled us to conclude that, in contrast to the digestibility of poly[d(A-T)] which yielded the expected 3'-mononucleotides, the polymerizing enzymes did not digest O4-methyl dT or its neighbors. The presence of the resistant alpha-phosphorothionate linkage did not prevent measurable digestion of poly[d(A-T)] by the Klenow fragment. This, together with evidence that polymerization of O4-methyl dTTP is favored at low temperatures, supports the model proposed by Ollis et al. [Nature 313, 762-766, 1985] showing independent domains for the two activities in the Klenow fragment.     

Effects of poly[d(pGpT).d(pApC)] and poly[d(pCpG).d(pCpG)] repeats on homologous recombination in somatic cells.

Sequencing studies have shown that in somatic cells alternating runs of purines and pyrimidines are frequently associated with recombination crossover points. To test whether such sequences actually promote recombination, we have examined the effects of poly[d(pGpT).d(pApC)] and poly[d(pCpG).d(pCpG)] repeats on a homologous recombination event. The parental molecule used in this study, pSVLD, is capable of generating wild-type simian virus 40 DNA via recombination across two 751-base-pair regions of homology and has been described previously (Miller et al., Proc. Natl. Acad. Sci. USA 81:7534-7538, 1984). Single inserts of either a poly[d(pGpT).d(pApC)] repeat or a poly[d(pCpG).d(pCpG)] repeat were positioned adjacent to one region of homology in such a way that the recombination product, wild-type simian virus 40 DNA, could be formed only by recombination within the homologies and not by recombination across the alternating purine-pyrimidine repeats. We have found that upon transfection of test DNAs into simian cells, a poly[d(pCpG).d(pCpG)] repeat enhanced homologous recombination 10- to 15-fold, whereas a poly[d(pGpT).d(pApC)] repeat had less effect. These results are discussed in terms of the features of these repeats that might be responsible for promoting homologous recombination.     

Stopped-flow kinetic analysis of the interaction of anthraquinone anticancer drugs with calf thymus DNA, poly[d(G-C)].poly[d(G-C)], and poly[d(A-T)].poly[d(A-T)]

The sodium dodecyl sulfate driven dissociation reactions of daunorubicin (1), mitoxantrone (2), ametantrone (3), and a related anthraquinone without hydroxyl groups on the ring or side chain (4) from calf thymus DNA, poly[d(G-C)]2, and poly[d(A-T)]2 have been investigated by stopped-flow kinetic methods. All four compounds exhibit biphasic dissociation reactions from their DNA complexes. Daunorubicin and mitoxantrone have similar dissociation rate constants that are lower than those for ametantrone and 4. The effect of temperature and ionic strength on both rate constants for each compound is similar. An analysis of the effects of salt on the two rate constants for daunorubicin and mitoxantrone suggests that both of these compounds bind to DNA through a mechanism that involves formation of an initial outside complex followed by intercalation. The daunorubicin dissociation results from both poly[d(G-C)]2 and poly[d(A-T)]2 can be fitted with a single exponential function, and the rate constants are quite close. The ametantrone and 4 polymer dissociation results can also be fitted with single exponential curves, but with these compounds the dissociation rate constants for the poly[d(G-C)]2 complexes are approximately 10 times lower than for the poly[d(A-T)]2 complexes. Mitoxantrone also has a much slower dissociation rate from poly[d(G-C)]2 than from poly[d(A-T)]2, but its dissociation from both polymers exhibits biphasic kinetics. Possible reasons for the biphasic behavior with the polymers, which is unique to mitoxantrone, are selective binding and dissociation from the alternating polymer intercalation sites and/or dual binding modes of the intercalator with both side chains in the same groove or with one side chain in each groove.     

Linear dichroism demonstrates that the bases in poly[d(AC)] · poly[d(GT)] and poly[d(AG)] · poly[d(CT)] are inclined from perpendicular to the helix axis

Flow linear dichroism is used to measure specific inclinations for each of the four bases in poly[d(AC)]·;poly[d(GT)] and poly[d(AG)]·poly[d(CT)] in both the B and A forms. For the B form in solution the bases are found to have a sizable inclination. Inclination is increased in the A form, as expected. In all cases the pyrimidines are more inclined than the purines. © 1993 John Wiley & Sons, Inc.     

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)].     

Mercury-induced transitions between right-handed and putative left-handed forms of poly[d(A-T).d(A-T)] and poly[d(G-C).d(G-C)]

Poly[d(A-T).d(A-T)] and poly[d(G-C).d(G-C)], each dissolved in 0.1 M NaClO4, 5 mM cacodylic acid buffer, pH 6.8, experience inversion of their circular dichroism (CD) spectrum subsequent to the addition of Hg(ClO4)2. Let r identical to [Hg(ClO4)2]added/[DNA-P]. The spectrum of the right-handed form of poly[d(A-T).d(A-T)] turns into that of a seemingly left-handed structure at r greater than or equal to 0.05 while a similar transition is noted with poly[d(G-C).(G-C)] at r greater than or equal to 0.12. The spectral changes are highly cooperative in the long-wavelength region above 250 nm. At r = 1.0, the spectra of the two polymers are more or less mirror images of their CD at r = 0. While most CD bands experience red-shifts upon the addition of Hg(ClO4)2, there are some that are blue-shifted. The CD changes are totally reversible when Hg(II) is removed from the nucleic acids by the addition of a strong complexing agent such as NaCN. This demonstrates that mercury keeps all base pairs in register.