Dynamics in the isomerization of intramolecular DNA triplexes in supercoiled plasmids.
We report here kinetic and thermodynamic studies on differential isomerization of intramolecular Pyr*Pur.Pyr triplexes in supercoiled plasmids. Two structural isomers of the triplex exist: one with the 3'-half of the Pyr strand as the third strand (H-y3 form) and the other with the 5'-half as the third strand (H-y5 form). The relative populations of the two triplex isomers was determined using the chemical probe with diethyl pryrocarbonate as a function of incubation time. The results demonstrated that triplexes were formed rapidly after a pH change from pH 8.0 to 5.0 and that the initial population of the two isomers exponentially changed with incubation time to reach true thermodynamic equilibrium with a time constant of 0.6-10 h, depending on temperature and the presence of Mg2+. The results clearly demonstrated that interconversion occurs between the two isomers and that the presence of Mg2+ generally retarded the interconversion rates. Kinetic and thermodynamic analyses of the relative populations of the two isomers revealed that the apparent energy barrier for transition from duplex to the H-y3 form is higher than that to the H-y5 form, but H-y3 is more stable in enthalpy terms than H-y5. Therefore, H-y3 is kinetically inferior but thermodynamically favored at higher supercoil levels in plasmids. The presence of Mg2+ resulted in both a kinetic and a thermodynamic preference for H-y5 formation, relative to the H-y3 form.
Shindo
H
H
School of Pharmacy, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-03, Japan. shindo@ps.toyaku.ac.jp
Matsumoto
N
N
Shimizu
M
M
D004247:DNA / Q000737:chemistry*; D004278:DNA, Superhelical; D004926:Escherichia coli; D006863:Hydrogen-Ion Concentration; D007536:Isomerism; D007700:Kinetics; D008274:Magnesium; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000737:chemistry*; D013816:Thermodynamics
D004278:DNA, Superhelical; C051802:triplex DNA; D004247:DNA; D008274:Magnesium