Biochemistry 1982, 21, 6567-6574 in the same way. In addition, when biological effects are considered in vivo, other selective effects must be evaluated experimentallysince it is well-known that reactions that appear to be kinetically inconsequential can produce amplifiable biological effects. While we make no claim that chain cleavage by 254-nm light is more important than pyrimidine dimer formation, or any other photoreaction, we do believe the reaction deserves further study both chemically and biologically. References Asteriadis, G. T., Armbruster, M. A., & Gilham, P. T. (1976) Anal. Biochem. 70, 64-74. Cerutti, P., Ikeda, K., & Witkop, B. (1965) J . Am. Chem. SOC. 87, 2505-2507. Chen, P. S., Jr., Toribara, T. Y., & Warner, H. (1956) Anal. Chem. 28, 1756-1758. Coahran, D. R., Buzzell, A., & Lauffer, M. A. (1962) Biochim. Biophys. Acta 55, 755-767. Goossen, J. T. H., & Kloosterboer, J. G. (1978) Photochem. Photobiol. 27, 703-708.
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Gordon, M. P., Huang, C., & Carter, J. (1976) in Photochemistry and Photobiology of Nucleic Acids (Wang, s. Y., Ed.) Vol. 11, pp 265-308, Academic Press, New York. Halmann, M., & Platzner, I. (1965) J . Chem. Soc., 5 380-5 38 5. Johns, H. E. (1969) Methods Enzymol. 16, 169-224. Rahn, R. O., & Patrick, M. H. (1976) in Photochemistry and Photobiology of Nucleic Acids (Wang, S . Y . , Ed.) Vol. 11, pp 98-105, Academic Press, New York, San Francisco, and London. Schulman, L. H., Kucan, I., Edelman, B., & Chambers, R. W. (1973) Biochemistry 12, 201-208. Tomasz, M., & Chambers, R. W. (1966) Biochemistry 5 , 773-7 8 2. Trachtman, M., & Halmann, M. (1977) J . Chem. Soc., Perkin Trans. 2, 132-137. Triantaphylides, C., & Halmann, M. (1975) J . Chem. Soc., Perkin Trans. 2, 34-40. Triantaphylides, C., & Gerster, R. (1977) J . Chem. Soc., Perkin Trans. 2, 1719-1724.
Kinetics for Exchange of Imino Protons in the d( C-G-C-G-A-A-T-T-C-G-C-G)Double Helix and in Two Similar Helices That Contain a G-T Base Pair, d( C-G-T-G-A-A-T-T-C-G-C-G),and an Extra Adenine, d( C-G-C-A-G-A-A-T-T-C-G-C-G)? Arthur Pardi,f Kathleen M. Morden, Dinshaw J. Patel,$ and Ignacio Tinoco, Jr.*
The relaxation lifetimes of imino protons from was a larger perturbation for opening of the base pairs than individual base pairs were measured in (I) a perfect helix, th? G-T base pair. The temperature dependence of the exd(C-G-C-G-A-A-T-T-C-G-C-G), (11) this helix with a G-C change rates of the imino proton in the perfect helix gives base pair replaced with a G-T base pair, d(C-G-T-G-A-A-Tvalues of 14-1 5 kcal/mol for activation energies of A-T imino T-C-G-C-G), and (111) the perfect helix with an extra adenine protons. These relaxation rates were shown to correspond to base in a mismatch, d(C-G-C-A-G-A-A-T-T-C-G-C-G). The exchange involving individual base pair opening in this helix, which means that one base-paired imino proton can exchange lifetimes were measured by saturation recovery proton nuclear magnetic resonance experiments performed on the imino independent of the others. For the other two helices that contain perturbations, much larger activation energies for protons of these duplexes. The measured lifetimes of the imino protons were shown to correspond to chemical exchange exchange of the imino protons were found, indicating that a lifetimes at higher temperatures and spin-lattice relaxation cooperative transition involving exchange of at least several base pairs was the exchange mechanism of the imino protons. times at lower temperatures. Comparison of the lifetimes in these duplexes showed that the destabilizing effect of the G-T The effects of a perturbation in a helix on the exchange rates and the mechanisms for exchange of imino protons from olbase pair in I1 affected the opening rate of only the nearestneighbor base pairs. For helix 111, the extra adenine affected igonucleotide helices are discussed. the opening rates of all the base pairs in the helix and thus ABSTRACT:
R e l a x a t i o n rates of the base-paired imino protons have been measured by proton nuclear magnetic resonance (NMR) ex+ From the Department of Chemistry and Laboratory of Chemical Biodynamics, University of California, Berkeley, California 94720. Received June 9, 1982. This work was supported by National Institutes of Health Grant GM 10840 and by the Division of Biomedical and Environmental Research of the Department of Energy under Contract No. 98 (DE-AC03-76SF00098). K.M.M. was supported by National Institute of Environmental Health Sciences Training Grant ES 07075. We also thank the Stanford Magnetic Resonance Laboratory (supported by National Science Foundation Grant GP 26633 and NIH Grant RR 0071 1) for the use of the HXS-360-MHz facilities. *Present address: Institut fur Molekularbiologie und Biophysik, Eidgenossiche Technische Hochschule, CH-8093 Zurich, Switzerland. *Present address: Bell Laboratories, Murray Hill, N J 07974.
0006-2960/82/0421-6567$01.25/0
periments in several nucleic acid systems (Crothers et al., 1974; Johnston & Redfield, 1977, 1978; Hurd & Reid, 1980; Early et al., 1981a,b). We recently studied the kinetics for exchange of imino protons in a DNA, RNA, and hybrid oligonucleotide helix (Pardi & Tinoco, 1982). The saturation recovery technique developed by Redfield (Johnston & Redfield, 1977) was used in these studies, and the theory for interpretation of the exchange behavior of imino protons measured by NMR has been discussed by Johnston & Redfield (1981) and Pardi & Tinoco (1982). The three helices used in this work, the 12-mer [helix I = d(C-G-C-G-A-A-T-T-C-G-C-G)], the 12-mer G-T [helix I1 = d(C-G-T-G-A-A-T-T-C-G-C-G)], and the 13-mer [helix I11 = d(C-G-C-A-G-A-A-T-T-C-G-C-G)] , have been studied 0 1982 American Chemical Society
6568
B I O C H E M IS T R Y
by 'H and 31PN M R (Patel et al., 1982a-c). The conformation and dynamics of these duplexes were observed by measuring the chemical shifts and nuclear Overhauser effects on the imino, base, and sugar protons. These studies demonstrated the existence of a G-T wobble base pair in helix I1 and showed that the extra adenine base in helix I11 was stacked in the helix (Patel et al., 1982b,c). The destabilizing influence of a G.T base pair or an extra adenine is reflected in the melting temperatures of these helices, which are approximately 57 and 52 OC in 0.1 M phosphate for the 13-mer and 12-mer G-T helices, compared to the -72 "C found for the 12-mer helix, under the same conditions. The N M R of these three helices, including preliminary reports of some of the work presented here, has recently been reviewed (Patel et al., 1982d). In this paper we have measured the relaxation rates of the imino protons in the 12-mer, 12-mer G-T, and the 13-mer using saturation recovery experiments. Activation energies for exchange of the imino protons were determined by measuring the temperature dependence of the lifetimes. The pH dependence of the relaxation rates of the imino protons in the 12-mer and 12-mer G-T was also measured. The lifetimes, the pH dependence of the lifetimes, and the activation energies for exchange of the imino protons allow the dynamics of these duplexes to be interpreted in terms of specific mechanisms for exchange of the imino protons. The results on the 12-mer G-T and 13-mer show that helix opening is important in exchange of these imino protons. The helix opening pathway is shown to be the dominant exchange mechanism in the DNA duplex, d(CA5G) + d(CT5G) (Pardi & Tinoco, 1982). For the 12-mer duplex the exchange of the imino protons takes place by an individual base pair opening mechanism. The effects of a G-T wobble base pair, and an extra adenine, on the opening rates of individual base pairs in the three helices are discussed, along with the general dynamics of base pair opening in doublehelical oligonucleotides. Materials and Methods The oligonucleotides were prepared by a modified triester method followed by deprotection and purification (Hirose et al., 1978; Patel et al., 1982a-c). The NMR experiments were performed on the HXS-360-MHz instrument at the Stanford Magnetic Resonance Laboratory, with the experimental methods described previously (Pardi & Tinoco, 1982). The NMR spectra were all run in 0.1 M phosphate buffer-2.5 mM ethylenediaminetetraacetic acid (EDTA) with the chemical shifts referenced to the internal standard sodium 4,4-dimethyl-4-silapentane- 1-sulfonate (DSS). The lifetimes were calculated, as previously discussed (Pardi & Tinoco, 1982), with no significant double-exponential behavior seen in any of the data. The lifetimes reported here are estimated to be accurate to within f20%. Typically, 10-15 different delay times were taken with 220-250 scans for each point. Some of the experiments discussed here were repeated on a 200-MHz instrument using methods similar to the long-pulse inversion recovery technique described by Early et al. (1980). The values for the lifetimes calculated from these experiments are not reported here but were found to be within experimental error of the reported values. Results 12-mer: d(C-G-C-G-A-A-T-T-C-G-C-G).Figure 1 shows a saturation recovery experiment performed on the 12-mer at 15 O C , pH 8. As discussed by Patel et al. (1982a), the terminal G.C base-paired imino proton was observed only at very low temperatures and so was not seen in the temperature range used in this study. The measured lifetimes of the other five
PARD1 ET A L . ~
~~
Table I: Lifetimes (ms) of Imino Protons in 12-mer at pH 6 1
2
3
4
5
6
6
5
4
3
2
1
d(C-G-C-G-A-A-T-T-C-G-C-G)
I J l l l l l l l l / /
(G-C-G-C-T-T-A-A-G-C-G-Cid
proton
temp
("C)
2
3
4
5
6
5 15 25 30 3s 40 45 50 55
150 140 100 90 40 30 18
260 205 140 175 130 80 60 50 35
230 255 190 240 170 160 150 80 80
250 360 310 230 140 125 90 65 35
270 320 350 280 235 200 130 90 55
Table 11: pH Dependence of Lifetimes (ms) of Imino Protons in 12-mer 1 2
3
4
5
6
6
5
4
3
d (C- G - C - G - A - A - T - T - C - G
2 ~
1
C -G )
/ / I /l l l l l l l l
(G-C- G-C - T - T -A-A-G-C-G-C)a
PH 6 8 6 8 6 8
proton
temp ("C)
15 35 45
2
3
4
5
6
140 160 40 23 18
205 230 130 170 60 b
255 260 170 280 150 180
360 275 140 180 90 70
320 280 235 235 130 105
a
Lifetime is difficult to measure a Too fast to measure,
