New DNA polymorphism: evidence for a low salt, left-handed form of poly(dG-m5dC)

volume 13 Number 11 1985 Nucleic Acids Research New DNA polymorphism: evidence for a low salt, left-handed form of poly(dG-m'dC) Burt G.Feuerstein1 + , Laurence J.Marton1-2, Max A.Keniry1, Donald L.Wade3 and Richard H.Shafer3' 'Brain Tumor Research Center, 'Department of Laboratory Medicine, School of Medicine, and 'Department of Pharmaceutical Chemistry, School of Pharmacy, University of California, San Francisco, CA 94143, USA Received 4 April 1985; Revised and Accepted 10 May 1985 IHIK0D0CTI0S The confirmation by X-ray crystal studies (1,2) of a left-handed form of poly(dG-dC) originally suggested by the work of Pohl and Jovin (3) has been a significant development In the understanding of nucleic acid structure (4,5). Recent experiments point to the likely biological importance of this flexibility in DNA conformation in terms of gene regulation (4,5). Various combinations of conditions and polynucleotides have been shown to result in a left-handed form (3-6). In the case of poly(dG-dC) and poly(dG-m dC), such conditions are either strongly dehydrating, Involving high salt concentrations or addition of alcohol, or require the presence of oligovalent cations such as spermlne (7). Furthermore, temperature also appears to play a role in the B to Z transition of both poly(dG-dC) and poly(dG-n5dC) (8,9). Wu and Behe (10) have recently described a low salt, Z conformation in the synthetic polymer poly(rG-dC). Upon increasing the salt concentration, this polymer undergoes a transition first to the A form and then to the Z form again. The conformation that Is stable at intermediate salt Is the A form, presumably due to the presence of an alternating rlbose-deoxyribose sugar backbone. In the experiments described below, we present evidence for a left- ©IRLPrew Limited, Oxford, England. 4133 ABSTRACT Spectroscoplc studies on solutions of poly(dG-tn dC) over a wide range of salt concentration are presented. Low salt solutions ([Na ]) < 2 mM) of poly(dG-m dC) produce circular dlchrolsm (CD) spectra typical of the lefthanded, Z form at high salt ([Na+] = 1.75 M ) . Solutions of poly(dG-m5dC) at Intermediate salt concentrations, e.g., 142 mM, yield CD spectra characteristic of the right-handed, B conformation. 3 1 P NMR spectra of the low salt form of poly(dG-m dC) reveal two well separated peaks, split by 1.4 ppm, consistent with a dinucleotide repeat. Kinetic studies show that the transition from the low salt form to the right-handed 8 form Is slow, as expected for a major conformations! change. These results suggest that the Z conformation in poly(dG-m dC) can be stabilized at very low salt as well as at high salt. Nucleic Acids Research handed form of the DNA polymer poly(dG-m dC) stable at very low salt concentrations in the absence of ollgovalent cations. Increasing concentrations of NaCl Lead first to the B conformation and then to the Z conformation. This unexpected result may have profound implications for our understanding of the forces that stabilize the various forms of DNA and adds another dimension to the possible biological role of DNA polymorphism. MATERIALS AMP METHODS Poly(dG-m dC) was obtained from Pharraacla-P.L. Biochemlcals. Concentrations of poly(dG-m dC) were determined spectroscoplcally using a When first 10 minutes. Samples for UV and CD (circular dichrolsm) studies were prepared by dialysis against low salt buffers (0.5 or 2 mM Na-cacodylate) containing 0.1 mM Na2EDTA. In some cases, stock solutions of poly(dG-m dC) were exhaustively dlalyzed against 2 M NaCl, 1 mM Na2EDTA, then dlalyzed back down to a low salt buffer. Samples for NMR studies were prepared by sonicating poly(dG-m5dC) in 0.5 mM Na-cacodylate, 1 M NaCl for 3 hr under C0 2 in an Iceethanol bath, followed by extensive dialysis first against 20 mM NH4HCO3, then against 5 mM NH^HCO-j (and 1 mM EGTA in each case) and lyophillzatlon. The low salt sample was made by resuspendlng the lyophillzed polynucleotlde In a buffer consisting of 0.5 mM Na-cacodylate, 0.1 mM Na2EDTA, 201 D 2 0, pH meter reading 7.4. The Intermediate salt solution was made by resuspendlng In H 2 0 containing 50 mM NaCl and 201 D 2 0, pH meter reading 7.5. Reproducible results were obtained on freshly prepared solutions or those stored at 4°C or -20°C for several days. Older solutions sometimes exhibited right-handed CD spectra under low salt conditions. UV spectra were recorded on a Cary 118 spectrophotometer while CD spectra were obtained on a JASCO 500A spectropolarimeter. 3 P NMR spectra were obtained on a home built multlnuclear spectrometer operating at 240 MHz for protons at the UCSF School of Pharmacy NMR Facility. Spectra were recorded at 45°C with quadrature detection, a sweep width of ± 630 Hz, a 65° pulse, a recycle time of 3.3s, 2W proton decoupling power and with 4K data points collected in each channel. trinethyl phosphate. Chemical shifts are presented relative to external 8000—9000 scans were accumulated and 25 Hz linebroadenlng was applied to each spectrum. The kinetics of the transition from both the low salt and high salt lefthanded conformations to the intermediate salt, B conformation were 4134 molar (base pair) extinction coefficient, e2gg, of 14,200. dissolving the polynucleotlde, the sample was typically annealed at 50°C for Nucleic Acids Research investigated as follows. A solution of poly(dG-m^dC) in 2 mM Na-cacodylate was mixed by hand with a solution of NaCl in the same buffer to yield a final NaCl concentration of 0.2 M. The CD amplitude at 292 nra was then monitored as a function of time after mixing. A similar experiment was then carried out 5 starting with poly(dG-m dC) in 2 mM Na-cacodylate and 1.75 M NaCl which was then mixed with enough buffer alone to bring the final NaCl concentration down to 0.2 M. In order to observe the bulk of the transition using hand mixing, these experiments were carried out at 16°C. RESULTS cacodylate are presented in Figure la. The high salt conditions produce an inverted spectrum typical of the Z form of this polymer. Intermediate salt conditions show a spectral pattern typical of the B form. Surprisingly, the low salt form displays a spectrum very similar to that of the high salt form, indicative of a left-handed conformation. Low concentrations of such multivalent cations as spermine and Mg"1"1* have been previously shown to induce the Z form in poly(dG-nrdC) (6). However, a conformatlonal change in poly(dGm-MC) at low sodium concentration in the absence of such multivalent cations has not been described previously. Figure lb shows the results of a NaCl tltration of the low salt form. The transition towards the right-handed form exhibits an isobestic point at 273 nm and occurs over a very narrow range of concentration, typical of a cooperative change in conformation (see Figure lc). Thus, as the Na + concentration increases from 2 mM or less, the CD spectrum changes from lefthanded t (...truncated)