Asymmetric opening reaction mechanism of Z-DNA base pairs: a hydrogen exchange study

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Abstract
With the tritium-Sephadex method, the hydrogen-exchange kinetics of the 5 NH protons of guanine and cytosine residues in Z-form poly(dG-dC) .cntdot. poly(dG-dC) were measured as a function of temperature and catalyst concentration. Over the measured temperature range from 0 to 34.degree. C, 2 classes of protons with constant amplitudes are found. The 3 protons of the fast class, which were assigned to the guanine amino and imino protons, have an exchange half-time in the minute time range (at 20.degree. C the half-time is 2.5 min) and an activation energy of 18 kcal mol-1. Since these 2 types of protons exchange at the same rate in spite of their grossly different pK values the exchange of these protons must be limited by the same nucleic acid conformational change. The 2 cytosine amino protons of the slow class are especially slow with exchange half-times in the hour time range (at 20.degree. C the exchange half-time is 1 h) and the activation energy is 20 kcal mol-1. The exchange of these 2 protons is not limited by some nucleic acid conformational change as shown by the marked exchange acceleration of these protons upon addition of 0.2 M imidazole. In addition, the hydrogen-deuterium exchange kinetics of the amino protons of guanosine cyclic 2'',3''-monophosphate were also examined by a spectral difference method using a stopped-flow spectrophotometer. The measured kinetic process is monophasic with a rate constant of 3 s-1 at 20.degree. C, which is in the same range as the predicted rate constant of the guanine amino protons. From the measurement of this exchange rate as a function of temperature between 10 and 35.degree. C, an apparent activation energy of 16 kcal mol-1 is found. The very peculiar dependence of the rate constant upon pH confirms the proposed general exchange mechanism that requires a preprotonation on the N7 position of guanine. A quantitative analysis of the Z-form poly(dG-dC) .cntdot. poly(dG-dC) hydrogen-exchange data with the classical 2-step exchange mechanism (closed .dblarw. open .fwdarw. exchanged) leads to the prediction of a non-opening limited exchange of the guanine amino protons, thus conflicting with the experimental data. Two extreme exchange mechanisms that can account for this set of data are discussed. The common feature of both mechanisms is the existence of an asymmetric open state where the guanine protons are more likely to be in contact with the solvent than the cytosine protons.