Conformations of 145 base pair length poly (dG-dC)·poly (dG-dC) in solution and in association with histones

Volume e Number 91980 Nucleic A c i d s Research Conformations of 145 base pair length poly (dG-dC) • poly (dG-dC) in solution and in association with histones Robert T. Simpson* and Heisaburo Shindo + Laboratory of Nutrition and Endocrinology* and Laboratory of Chemical Physics+, National Institute of Arthritis, Metabolism and Digestive Diseases, National Institutes of Health, Bethesda, MD 20205, USA Received 22 January 1980 ABSTRACT We have studied the conformation of poly(dG-dC)-poly(dG-dC) in three conditions; i) associated with histane octamers, ii) alone at ionic strength 0.1, and iii) in solutions of over 2.5 M NaCl. The circular dichroism spectrum for the polymer bound to histones differs from that for the free polymer; the difference spectrum is similar to those for native and Doly(dA-dT)poly(dA-dT)core particles. Under the first two conditions, the " P M4R spectrum is symmetric with line widths of 91 and 41 Hz, respectively, at 109.3 NHz. In high salt, two " p peaks of equal intensity are observed, confirming recent results of Patel et al. (1) and indicating an alternating geometry for the phosphodiester backbone. Using this highly homogeneous DNA, we confirm that the Pohl-Jovin transition (2) is an intramolecular rearrangement, not requiring complete strand separation. INTRODUCTION Synthetic polydeoxyribonucleotides offer a unique advantage to those interested in nucleic acid structure; the synthetic molecules amplify features of a particular sequence, often enabling detection of structures enforced on the nucleic acid by such sequence (for review, see 3). The structural features may be masked in sequence heterogeneous native TJNA, which probably has the entire spectrum of such allowable conformations. Two recently presented examples of such experimental virtues of these synthetic molecules follow. Using P W R , we have been able to detect an alternating conformation for the phosphodiester backbone of poly(dA-dT)poly(dA-dT) both in solution and in the fiber state (4,5) and also, Patel and Canuel noted sequence-dependent variations in phosphodiester torsion angles for the octanucleotide dG-dG-dA-dA-dT-dT-dC-dC duplex in solution (6). Furthermore, Patel et al. noted an alternating B-TJNA conformation for short poly(dG-dC) oligomers under high salt conditions (1), where the Pohl-Jovin transition (2) had occurred. These experimental data support a proposal offered at about the same time by Klug et al. (7) that such © IRL Press Umfttd. 1 Falconberg Court, London W1V6FG, U.K. 2093 Nucleic Acids Research alternating conformations might exist for alternating deoxypurine-deoxypyriraidine polymers. Second, we and others have studied semi-synthetic chromatin core particles formed from the inner histone octamer and poly(dA-dT)-poly(dA-dT) (8-10) or poly(dG-dC)-poly(dG-dC) (8). Particularly for the particles containing the AT polymer, removal of the effects of DNA sequence heterogeneity on physical and biochemical properties enabled refinement in the resolution of experiments on nucleic acid-protein interactions in these chromosomal particles (8-12). We have previously reported results of studies of the conformation of 145 bp poly(dA-dT)• poly(dA-dT) both in solution and in the core particle, using several spectroscopic methods (4,8,11,12). Here we describe analogous investigations of polyfdG-dC)-poly(dG-dC) structure. EXPERIMENTAL SECTION Poly(dA-dT).poly(dA-dT) and poly(dG-dC)-poly(dG-dC) were products of P-L Biochemicals. Chicken erythrocyte inner histones (H2A, H2B, H3 and H4) were prepared as previously described and associated with the synthetic polynucleotides by salt step dialysis (8). Digestions with micrococcal nuclease (Worthington Biochemical Corp.) and fractionation of digests by isokinetic sucrose aradient centrifugation were done exactly as before (8). DNA was isolated from core particle preparations by phenol extraction of a solution containing It sodium dodecyl sulfate and 25 mM EDTA, pH 7, and precipitation at -20° with 2.5 volumes ethanol. Conditions and methods for measurement of circular dichroism (16) and P M«IR (4,12) spectra have been detailed previously. Electrophoresis of DNA samples was performed on 5t polyacrylamide gels using a tris/borate/ 2094 A serendipitous offshoot of these latter experiments has been the ability to produce, by nuclease digestion of such semi-synthetic chromatins, highly homogeneous preparations of the alternating copolymers in double stranded form with length about 145 base pairs (bp). High molecular weight synthetic DNA is readily available from unprimed, ENA polymerase I-mediated synthesis (13,14) and small oligomers can be obtained synthetically or by fractionation of partial enzymatic digests of higher molecular weight material (15); preparations from semi-synthetic chromatins fill a gap between these two size groups. This size ENA has advantages for several types of physical study of structure in being small enough to tumble rapidly, enhancing resolution in NMR experiments, yet large enough that end effects on properties are expected to be small. Nucleic Acids Research EDTA buffer system (17). Approximate DNA sizes are based on calibrations of the gel using the mobilities of bromphenol blue and xylene cyanol FF (18). RESULTS AND DISCUSSION Circular Dichroism Spectra Figure 1 shows the CD spectra for core particles containing poly(dG-dC)poly(dG-dC) and the 14S base pair polynucleotide in the absence of proteins. The core particle spectrum is dissimilar to both that of native core particles (19) and that of nucleosomes containing poly(dA-dT)-poly(dA-dT) (9,11). A broad positive ellipticity band with ™ H I M at 298 ran (800°) and 275 ran 240 260 280 WAVELENGTH 300 Figure 1 Circular dichroism spectra of 145 bp poly(dG-dC)-poly(dG-dC). Spectra were determined for the synthetic polynucleotide associated with inner histones as a semi-synthetic core particle ( — ) , alone in solution at ionic strength 0.1M (---), and alone in solution in approximately 3 M Nad ( ) . Inset: the difference CD spectrum for the core particle polynucleotide at ionic strength 0.1 M. 2095 Nucleic Acids Research P Nuclear Magnetic Resonance Spectra The P NMR spectra of core particles containing poly(dG-dC)-poly(dGdC) and the protein-free nucleic acid were recorded at 20° (not shown). The two spectra are symmetrical and fit well by single Lorenzian distributions. Chemical shifts are virtually identical for phosphorus in either case. The spectra do differ in line width, 41 Hz for the DNA and 91 Hz for the nucleoprotein particle at an observing frequency of 109.3 htiz. Line width for the GC-containing particle is significantly less than that for native particles with random sequence DNA, 130 Hz (21), or semi-synthetic particles containing poly(dA-dT)-poly(dA-dT), 110 Hz (12). The differences between the line width for the GC-containing DNA and the same when in a core particle (91-41-50 Hz) and between the overall line width (...truncated)