Oct 15, 2018 - base-paired structure is wobble, whereas at higher pH an ionized ..... data on an enol tautomer structure has been reported. 2) The rare ...
THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1988 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 263, No. 29, Issue of October 15, pp. 14794-14801,1988 Printed in U.S.A.
Equilibrium between a Wobble and Ionized Base PairFormed between Fluorouracil and Guanine in DNA as Studied by Proton and Fluorine NMR* (Received for publication, September 1, 1987)
Lawrence C. SowersS1, Ramon Eritjag,Bruce Kaplans, MyronF. Goodmany, and G. Victor Fazakerlyll From the (Molecular Biology Section, Department of Biological Sciences, University of Southern California, Los Angeles, California 90089-1340, the §Department of Molecular Genetics, BeckmanResearch Institute of the City of Hope, Duarte, California 91010, and the IIService de Biochimie, Departement de Biologie, Centre d’Etudes Nucleaires de Saclay, 91191 Gif-sur- Yvette Ceden, France
A synthetic oligonucleotide duplex containing the chemotherapeutic and mutagenic agent 5-fluorouracil paired with guanine has been studied in solution by proton and fluorine NMR. The 7-mer duplex containing a central FU-G base pair adopts a normal righthanded configuration. At low pH, the predominant base-paired structure is wobble, whereas at higher pH an ionized structure in Watson-Crick geometry i s observed. The two structures are in a pH-dependent equilibrium with one another with an apparent pK of 8.3 at 23 “C. This is the first demonstration of an equilibrium between two distinct base pairing schemes and the first demonstration of a negatively charged base pair in DNA.
Fluorouracil and itsderivatives comprise an importantclass of chemotherapeutic agents (1, 2). In addition to their well known toxicity, these compounds are also mutagenic (3) and oncogenic (4). Fluorouracil derivatives are known to be incorporated into DNA both in vitro (5, 6) and in vivo (6-12). Transition mutations induced by fluorouracil could presumably arise as a consequence of the formation of fluorouracilguanine base pairs, in analogy with the mutagenic mechanisms demonstrated with bromouracil (13-15). To date, three possible structures have been proposed for uracil and substituteduracil base pairs with guanine: wobble (16-23), rare tautomer (13,24-28),and ionized forms (13,2933). Since the free energy for ionization of FU’ is close to zero at physiological pH (33-35), FU is an ideal candidatefor examination of a possible pH-dependent equilibrium between geometrically distinct structures. EXPERIMENTALPROCEDURES
FdU was obtained from Sigma and converted to its 5”dimethoxytrityl derivative according to Narang et al. (36). The 3’-phosphotriester was prepared according to Yamada and Dohmori (37). The two oligonucleotides shown below were prepared by standard phospho-
* This work was supported by Grants GM 33863 and GM 21422 and Biomedical Research Support Grant 2S07RR05471from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solelyto indicate this fact. $ Present address: Division of Pediatrics, City of Hope National Medical Center, Duarte, CA 91010. The abbreviations used are: FU, 5-fluorouracil; FdU, 5-fluoro-2’deoxyuridine; NOE, nuclear Overhauser effect; NOESY, two-dimensional nuclear Overhauser effect spectroscopy; FUrd, 5-fluouridine.
triester methods except that the strand containing the fluorouracil residue (FU) was deprotected in concentrated ammonium hydroxide for 3 days at 37 “C rather than overnight at 60 “C. 5’ d (C 1 3’ d (G 14
A 2 T 13
G 3 C 12
FU 4 G 11
G 5
C 10
G 6 C 9
C) strand 1 7 G ) strand 2 8
Samples were prepared in 10 mM phosphate buffer (pH 7.2) (unless otherwise stated), 150 mM NaCl, and 0.2 mM EDTA, at a strand concentration of 4 mM. The two strands were annealed by heating to 80 “Cfollowed by slow cooling. NMR spectra were recorded in either 99.996% DzO or 90% H20, 10% D20. Proton chemical shifts were measured relative to internal tetramethylammonium chloride, 3.18 ppm. NMR spectra were recorded on a Bruker WM-500 spectrometer. Two-dimensional NOESY spectra were recorded in the phase-sensitive mode (38) with 2K data points in the t2 dimension and 150-250 acquisitions/spectrum. 250 free induction decays were collected in the tl dimension with mixing or 40 ms. The residual HDO (hydrogen times of250,200, deuterium oxide) resonance was weakly presaturated during the relaxation decay. After zero filling to give a 2K x 2K matrix, a slightly shifted sinebel1 function was applied to the data in both dimensions prior to Fourier transformation for figure presentation. In addition, the 40-ms spectrum was treated with a 90” sine bell function in bothdimensions before Fourier transformation. This spectrum was used for the measurement of cross-peak volumes. Spectra in 90% Hz0 were recorded using a 1-7-1hard pulse sequence (39). Fluorine NMR spectra were recorded at 282 MHz ona Bruker MSL-300 spectrometer. ”F resonance chemical shifts were measured relative to external trifluoroacetic acid. RESULTS
Neutral p H Exchangeable Protons-The resonances of the exchangeable protons, those involved in hydrogen bonding and base pair formation, were examined in 90% HzO. The one-dimensional spectrum of the duplex recorded at 1“C and pH 7.2 is shown in Fig. la. We observe, in the low field part of the spectrum, one resonance in the usual thymidine region at 14.12 ppm. In the guanosine imino proton region, 12.513.5 ppm, we observe three resonances of which the one at 13.01 ppm integrates for three protons. The remaining two resonances, at 12.23 and 10.52 ppm, most probably arise from the FU.G base pair. Resonances in this region of the spectrum
14794
NMR PairFluorouracil-Guanine Base
~
c10
,
n G11
FIG. 1. a, spectrum in 90% H20 (pH 7.2) a t 1 "C. b, c and e, difference spectra following 0.5-s selective presaturation of an imino resonance as indicated. d as c except that thepH was 6.0. f , spectrum of the duplex at pH 9.5, 1 "C.
14795
one at 12.23 ppm must be an NOE within the FU.G base pair. We note that this latter NOE is no larger than the interresidue NOEs, which may appear unusual. However, the reference spectrum, (Fig. la) shows that the line widths of of the the two high fieldimino protons are greater than those other resolved imino resonances (see also below). This indicates the presence of an exchange phenomenon which may reduce the magnitude of the NOE between the resonances a t 12.23 and 10.52 ppm. Note the verylarge NOE to an exchangeableprotonresonance at 6.05 ppm. While cytidine nWC amino proton resonances are found in theregion of 67 ppm (45, 46), themagnitude of thisNOE excludes an interresidue NOE, and, in anycase, no corresponding intense WC amino resonance is observed in the region of 8-9 ppm. Furthermore, all the cytidine amino resonances have been assigned (see below) and for the nWC amino protons these are found in the region of 6.4-7.1 ppm. It must therefore be attributed to the G amino group of the FU .G base pair in rapid rotation ona proton NMR time scale. two NOEs Presaturation at12.23 ppm gives rise to the same in theguanosine imino protonregion (Fig. IC)and thereverse NOE at 10.52 ppm. In the aromaticregion, small interresidue NOEs to cytidine amino protons observed are and also aweak NOE at 6.05 ppm. While we cannot decrease the temperature, we observe that at pH 6.0 the line widthsof the two high field resonances decrease. Repeating the experimentshown in Fig. ICat pH 6.0 (Fig. Id), shows that the magnitudeof the NOE between the resonances at 12.23 and 10.52 ppmincreases relative to those of the interbase pair NOEs. This confirms that the apparently small NOE observed at pH 7.2 results from a n exchange phenomenon. Presaturation of one of the guanosine imino resonances seen in Fig. 1, b and c), at 13.37 ppm results ina normal NOE to a guanosine imino proton at13.13 ppm but only very weak NOEs to the two high field imino resonances (Fig. le). The observed differences in the NOE magnitudes, again, indicate G pair) is exchangethat the relaxationof these protons (FU. influenced at pH 7.2. We observe large NOEs to the amino group of C10, weaker NOEs to the adjacentC9 amino group and a small NOE to the G amino group of the FU.G base pair. Note that we do not observe an NOE which can be attributed to the G5 amino protons and is this the casefor all G . C base pairs. At this temperature we expect that these resonances are very broad (45). Hydrogen bonding in G . C base pairs slows down rotation of the G amino group to intermediate exchange on a proton time scale. From presaturation of thethymidineiminoresonance(notshown)the complete resonance assignment canbe made as shown in Table I. T o further confirm assignment of the resonance at 6.05 ppm, spectra were recorded in D20 and H20at 12 "C under identical experimental conditions. For these experiments, the origin of the spectrumwas set at6.8 ppm. Comparing thetwo spectra the resonance corresponding to that at 6.05 ppm is
have been observed either for G. T base pairs (17-20) or loop TABLEI structures (40-44). Chemical shifts of the exchangeable protons at I "C (pH 7.2) Resonance assignment was carried outby standard presatCytidine uration techniques. The spectral resolution in the imino reGuanosine amino Imino pairs Base amino gion is such that the assignment is readily obtained by onewc nWC dimensional spectra. Furthermore,some of the NOEs are very 8.25 7.02 case G14 C1. weak or very close to the solvent resonance, in which13.02 (7.78, 14.12 A(H2)) one-dimensionalspectracan give betterresultsthan two- T13A2. G3.Cl2 13.02 8.20 6.86 dimensional spectra with solvent suppression. Presaturation FU4.Gl1 12.23, 10.52 6.05 for 0.5 s of the resonance at 10.52 ppm gives rise to three G5. C10 13.37 8.55 6.92 G6. C9 8.23 6.45 13.13 NOEs in the low field region (Fig. l b ) , two of which are C7. G8 8.20 13.02 6.47 certainly the adjacent guanosine imino protons. The third
NMR Fluorouracil-GuanineBase Pair
14796
definitely an exchangeable resonance, is well resolved from PP the anomeric proton resonances, and integrates for two protons (data notshown). 5.: We have recorded spectra at pH 7.2 as a function of temperature. Imino proton line widths can give an estimate of the proton exchange rate with the solvent if exchange is in the open limited case. This canbe investigated by varying the phosphate concentration and pH. For reasons discussed below, we have only varied the buffer concentration from 10 to 50 mM, recording spectra at 20 “C. We observed a small increase in line width for the thymidine imino proton and 5 . ; also for the terminal guanosine imino protons. The remaining resonances showno change; however, this observation is probably not sufficient to establish whether exchange is in -c12 “13 I the open limited case, and thus we can only interpret the ! imino line widths qualitatively. We observe even at 1 “C that the line widths of the two imino proton resonances of the FU .G base pair arelarger than those of the other nonterminal base pair resonances (Fig. 2). Due to resonance overlap at 13.01 ppm the line width of the G3 imino cannot be measured 6.1 over the entire temperature range but only when the resonances of the terminal base pairs have disappeared. I I 1 I 1 We observe (Fig. 2) that the line widths of the FU.G base 8.1 7.7 7.3 PPm pair resonances increase on raising the temperature from 1 “C, FIG.3. Expanded NOESY contour plot of the cross-peaks while those of all the other nonterminal base pair resonances between the HS-HB region and the Hl’-HS region. The intense remain unchanged or decrease slightly as the temperature is peaks (x) arise from C(H5)-(H6)connectivities. raised to -15 “C. Only abovethis temperature does exchange contribute to the line width for these latter resonances. Neutral pH, Nonexchangeable Protons-The strategy for r the sequential assignment of nonexchangeable proton resonances has been described in detail (47-50) and will not be P P ‘ repeated here. Fig. 3 shows the region of the NOESY spectrum recorded with a mixing time of 250 ms at 20 “Cfor interactions 1.8. between base H8-H6 protons and the anomeric and cytidine H5 protons. The sequential connectivities can be started from the 5”terminal C1 residue, the H6 of which is found at 7.54 ppm. The connectivity of A2 is straightforeward, after which the chain can befollowedby reference to the interresidue
L
2.2
2.6
3.0
FIG.4. Expanded NOESY contour plot for HS-H6 to H2’H2”, T(CHs)connectivities. Intraresidue NOESare linked by solid lines and interresidue NOEs by broken lines.
0
10
20
.,
30
OC
1
FIG. 2. Line widths of the nonterminal imino proton resonances at pH 7.2 asa function of temperature. G3;A, G5;0, G6;A, T13;0, G11;and 0, FU4.
cross-peaks in the base proton H2’,2” region (Fig. 4). These connectivities lead to assignment of the resonance at 7.57 ppm to the H6 of FU. This resonance is a doublet of -7-Hz coupling in the resolution enhanced one-dimensional spectrum. The connectivities for strand B can also be followed,as shown. The interresidue NOEs to the H2’,H2” protons of C12 and T13 are readily identified in Fig. 4, and this allows assignment of the respective anomeric proton resonances from examination of the Hl‘-H2’,H2” region of the NOESY spectrum (not shown). Three interbase cross-peaks are ob-
NMR Fluorouracil-Guanine Base servedG8(H8)-C9(H5),Gll(H8)-C12(H5),and Cg(H6)ClO(H5). These are labeled B-D, respectively, in Fig. 3. The cross-peak intensity ratios observed in Figs. 3 and 4 are typical of a normal right-handed B helix and do not indicate any major distortion at or adjacent to theFU .G base pair. Geometric distortions could, however, bemasked by spin diffusion effects which are far from negligible at a mixing time of250 ms.We therefore recorded another NOESY spectrum with a mixing time of 40 ms andmeasured the crosspeak volumes in this spectrum (see "Experimental Procedures"). At this short mixing time the ratio 3f the volumes for the interresidue NOEs between a base H8-H6 proton and its H2' relative to that observed on the H2" is in the range 10-20. This ratio is what would be expected for a normal B DNA and shows the absence of spin diffusion at this short mixing time as has recently been shown (51). Comparing the intra- and interresidue NOEs through A2G6 and C9-Tl3 with those of the same type of interactions outside the central trinucleotide, no significant differences are discernible going through the mismatch site. The small variations are within the range normally observed for a sequence containing only Watson-Crick base pairs. Examination of all other regions of the 40-ms spectrum fail to establish the orientation of the FU .G base pair. This is not unexpected, as we have previysly shown (20) that the short interproton distances (
