Effect of Mg2plus on kinetics of oxidation of pyrimidines in duplex DNA by potassium permanganate

Po tas sium per manga nate ox i da tion of py rim i dine bases is of ten used to probe sin­gle-stranded re gions in func tional DNA-pro tein com plexes. How ever, so far re ac tiv- ity 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 quan ti ta tive KMnO4 footprinting, in con nec tion with par al lel stud ies on the ef fect of Mg2+ on ki net ics of ox i da tion of in di vid ual thymines in the sin­gle-stranded re gion of the open tran scrip tion com plex of Escherichiacoli RNA poly­mer ase at a cog nate Pa pro moter con tained in this plasmid. Rate con stants of ox i da­tion for pyrimidines, kj, in selected regions of pDS3 DNA, including Pa promoter, were de ter mined un der sin gle-hit re ac tion con di tions in the ab sence and pres ence of 10 mM MgCl2. Their val ues ap peared to be se quence-dependent and were: (i) the larg­est for Ts in 5 TA3' and 5 TC3' steps, while 2-4 times smaller for 5'-adja cent ones in TT(A,G,C) and TTT(A) runs, (ii) for Cs in 5'TC3' steps 2-4 fold smaller than for ad ja- cent Ts, and (iii) in the pres ence of Mg2+ gen er ally larger by a se quence-dependent fac­tor: in 5' TC3' steps of about 2 and 4 for Ts and Cs, re spec tively, in 5 TA3' steps of TTA and TTTA se quences for 3 -ter mi nal Ts of about 3, while for their 5 -neigh bors of a dis­tinctly smaller value of about 2. Com par i son of kj data for cor re spond ing Ts lo cated be tween +1 and -10 re gions of Pa pro moter in dsDNA and in ssDNA form in the open transcription complex, reported elsewhere, demonstrates that reactivity of pyrimi­dines in dsDNA is by 2-3 or ders of mag ni tude smaller. The ef fect of Mg 2+ in dsDNA is in ter preted in terms of elec tro static bar rier to dif fu sion of MnO4- on DNA surface, which is low ered by dif fu sive bind ing of these ions to back bone phos phates, in volv ing also se quence-specific con tacts with bases in the mi nor and major grooves of B-DNA.