Effect of Mg2+ on kinetics of oxidation of pyrimidines in duplex DNA by potassium permanganate.

Abstract

Potassium permanganate oxidation of pyrimidine bases is often used to probe single-stranded regions in functional DNA-protein complexes. However, so far reactivity of these bases in double-stranded DNA has not been studied quantitatively. We have investigated the kinetics of oxidation of pyrimidines in supercoiled pDS3 plasmid dsDNA by quantitative KMnO4 footprinting, in connection with parallel studies on the effect of Mg2+ on kinetics of oxidation of individual thymines in the single-stranded region of the open transcription complex of Escherichia coli RNA polymerase at a cognate Pa promoter contained in this plasmid. Rate constants of oxidation for pyrimidines, kj, in selected regions of pDS3 DNA, including Pa promoter, were determined under single-hit reaction conditions in the absence and presence of 10 mM MgCl2. Their values appeared to be sequence-dependent and were: (i) the largest for Ts in 5'TA3' and 5'TC3' steps, while 2-4 times smaller for 5'-adjacent ones in TT(A,G,C) and TTT(A) runs, (ii) for Cs in 5'TC3' steps 2-4 fold smaller than for adjacent Ts, and (iii) in the presence of Mg2+ generally larger by a sequence-dependent factor: in 5'TC3' steps of about 2 and 4 for Ts and Cs, respectively, in 5'TA3' steps of TTA and TTTA sequences for 3'-terminal Ts of about 3, while for their 5'-neighbors o tinctly smaller value of about 2. Comparison of kj data for corresponding Ts located between +1 and -10 regions of Pa promoter in dsDNA and in ssDNA form in the open transcription complex, reported elsewhere, demonstrates that reactivity of pyrimidines in dsDNA is by 2-3 orders of magnitude smaller. The effect of Mg2+ in dsDNA is interpreted in terms of electrostatic barrier to diffusion of MnO4- on DNA surface, which is lowered by diffusive binding of these ions to backbone phosphates, involving also sequence-specific contacts with bases in the minor and major grooves of B-DNA.