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2 Acta Biochimica Polonica

2 2001

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

100%

Łoziński T. , Wierzchowski K. L.

tom 48

nr 2

511-523

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 of a distinctly 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.

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

84%

Lozinski T. , Wierzchowski K. L.

Acta Biochimica Polonica

2001

tom 48

nr 2

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.