Supercoil-stabilized intramolecular triplexes have been described under various conditions in different polypurine.polypyrimidine sequences such as (dG)n.(dC)n and mixed sequences including d(GA)n.d(CT)n while information about the triplexes in (dA)n.(dT)n is scarce. Using osmium tetroxide complexes and diethyl pyrocarbonate as structural probes, we show a pyrimidine.purine.pyrimidine (TAT) triplex in (dA)58.(dT)58 sequence in a supercoiled plasmid pE19. Strong modification of approximately six central thymines and approximately six T's at the 3'-end of the (dT)58 stretch as well as the DEPC modification of the 5'-half of the (dA)58 strand suggested the prevalence of the H-y3 triplex conformer. At native superhelix density, optimum conditions for the triplex formation were close to 1 mM MgCl2, pH 8.5. At room temperature and MgCl2 concentrations below 0.5 and above 5 mM, almost no triplex was formed. It is suggested that the absence of the triplex at higher MgCl2 concentrations is due to the stabilization of the duplex by Mg2+ ions which prevents the duplex opening necessary for the triplex formation. At higher temperatures, favorable for duplex opening (e.g. 55 degrees C), the TAT triplex is formed even in the presence of 10 mM MgCl2. Among Ca2+, Sr2+, Ba2+, Cd2+, Zn2+ and Ni2+, only Ca2+ and Sr2+ yielded a modification pattern similar to that obtained with Mg2+; the modification pattern produced in the presence of Sr2+ was, however, much less intense. In the presence of 1 mM MgCl2, a decrease in pH from 8.5 to 7.7 resulted in a strong decrease of the triplex content. At highly negative superhelix density, the conditions for triplex formation were less stringent, and the triplex was observed even in the absence of MgCl2.

Institute of Biophysics Academy of Sciences of the Czech Republic Brno Czech Republic

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p53 Specifically Binds Triplex DNA In Vitro and in Cells