Structural characterization of separated H DNA conformers.

Polypyrimidine/polypurine DNA sequences in plasmids can adopt protonated triplex-containing structures (H DNA) in response to negative superhelical stress and low pH. A d(TC)17-d(GA)17 insert adopts two isomeric protonated structures, which differ in degree of helical unwinding. The variant forms of individual topoisomers were separated by agarose gel electrophoresis and their reactivities to permanganate and acid-induced depurination were compared. Depurination patterns of the individual conformers indicate that in the more mobile form (H-y5) the 5'-half of the d(GA)n strand participates in a triplex while in the other (H-y3) the 3'-half forms the triplex. The H-y5 form is more stable than the H-y3 form at low negative superhelix densities. Because of the difference in helical unwinding, the H-y5 form becomes relatively less stable as the superhelix density increases. Topological models of the two forms show that providing there is no linkage at the tips of the triple helical segments one more positive twist is localized in the H-y5 form than in the H-y3 form. The foldback in the pyrimidine strand of the H-y5 form is less accessible to solvent than that of the H-y3 form as assessed by its lower reactivity to permanganate. Consideration of a pyrimidine loop model (Harvey, S. C., Luo, J., & Lavery, R. (1988) Nucleic Acids Res. 16, 11795-11809) suggests that the unique stability of the H-y5 form results from Watson-Crick base pairs between residues of the d(TC)n loop and the d(GA)n strand as it exits the triplex.