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Zn2+ selectively stabilizes FdU-substituted DNA through a unique major groove binding motif.

Ghosh S, Salsbury FR, Horita DA, Gmeiner WH - Nucleic Acids Res. (2011)

Bottom Line: Mg(2+) neither inhibited EtBr complexation nor had as strong of a stabilizing effect.DNA sequences that did not contain consecutive FdU were not stabilized by Zn(2+).A lipofectamine preparation of the Zn(2+)-DNA complex displayed enhanced cytotoxicity toward prostate cancer cells relative to the individual components prepared as lipofectamine complexes indicating the potential utility of Zn(2+)-DNA complexes for cancer treatment.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Biology, Program in Molecular Genetics, Wake Forest University School of Medicine, Winston-Salem, NC 27157, USA.

ABSTRACT
We report, based on semi-empirical calculations, that Zn(2+) binds duplex DNA containing consecutive FdU-dA base pairs in the major groove with distorted trigonal bipyramidal geometry. In this previously uncharacterized binding motif, O4 and F5 on consecutive FdU are axial ligands while three water molecules complete the coordination sphere. NMR spectroscopy confirmed Zn(2+) complexation occurred with maintenance of base pairing while a slight hypsochromic shift in circular dichroism (CD) spectra indicated moderate structural distortion relative to B-form DNA. Zn(2+) complexation inhibited ethidium bromide (EtBr) intercalation and stabilized FdU-substituted duplex DNA (ΔT(m) > 15 °C). Mg(2+) neither inhibited EtBr complexation nor had as strong of a stabilizing effect. DNA sequences that did not contain consecutive FdU were not stabilized by Zn(2+). A lipofectamine preparation of the Zn(2+)-DNA complex displayed enhanced cytotoxicity toward prostate cancer cells relative to the individual components prepared as lipofectamine complexes indicating the potential utility of Zn(2+)-DNA complexes for cancer treatment.

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Zn2+, but not Mg2+, inhibits EtBr intercalation into duplex DNA containing consecutive FdU–dA base pairs. EtBr exclusion data are shown for each of the four DNA hairpins in the presence of the indicated concentrations of Zn2+ (red) or Mg2+ (blue). (A) 3′FdU hairpin; (B) 5′ FdU hairpin; (C) Alt-FdU hairpin; (D) No-FdU sequence.
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Figure 3: Zn2+, but not Mg2+, inhibits EtBr intercalation into duplex DNA containing consecutive FdU–dA base pairs. EtBr exclusion data are shown for each of the four DNA hairpins in the presence of the indicated concentrations of Zn2+ (red) or Mg2+ (blue). (A) 3′FdU hairpin; (B) 5′ FdU hairpin; (C) Alt-FdU hairpin; (D) No-FdU sequence.

Mentions: The formation of specific complexes for the four DNA hairpin sequences with Zn2+ and with Mg2+ was evaluated by measuring the quenching of EtBr fluorescence. The results are summarized in Figure 3. Addition of Mg2+ resulted in a slight, concentration-dependent decrease in EtBr fluorescence for all DNA hairpins except Alt–FdU (∼85% of control). In contrast, the effects of Zn2+ were highly sequence dependent. The effects of Zn2+ on the control sequence were similar to the effects of Mg2+ with a moderate concentration-dependent decrease in EtBr fluorescence. The effect of Zn2+ on the ‘Alt–FdU’ sequence was also moderate. In contrast, Zn2+ had a dramatic effect on EtBr fluorescence for the 3′-FdU and 5′-FdU sequences. Addition of 1 mM Zn2+ resulted in attenuation of EtBr fluorescence to <25% of control for these two sequences (Figure 3). The results are consistent with Zn2+ forming specific complexes with DNA sequences containing consecutive FdU nucleotides. Similar complexes do not form with Mg2+ or with sequences containing non-consecutive FdU nucleotides.Figure 3.


Zn2+ selectively stabilizes FdU-substituted DNA through a unique major groove binding motif.

Ghosh S, Salsbury FR, Horita DA, Gmeiner WH - Nucleic Acids Res. (2011)

Zn2+, but not Mg2+, inhibits EtBr intercalation into duplex DNA containing consecutive FdU–dA base pairs. EtBr exclusion data are shown for each of the four DNA hairpins in the presence of the indicated concentrations of Zn2+ (red) or Mg2+ (blue). (A) 3′FdU hairpin; (B) 5′ FdU hairpin; (C) Alt-FdU hairpin; (D) No-FdU sequence.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3105383&req=5

Figure 3: Zn2+, but not Mg2+, inhibits EtBr intercalation into duplex DNA containing consecutive FdU–dA base pairs. EtBr exclusion data are shown for each of the four DNA hairpins in the presence of the indicated concentrations of Zn2+ (red) or Mg2+ (blue). (A) 3′FdU hairpin; (B) 5′ FdU hairpin; (C) Alt-FdU hairpin; (D) No-FdU sequence.
Mentions: The formation of specific complexes for the four DNA hairpin sequences with Zn2+ and with Mg2+ was evaluated by measuring the quenching of EtBr fluorescence. The results are summarized in Figure 3. Addition of Mg2+ resulted in a slight, concentration-dependent decrease in EtBr fluorescence for all DNA hairpins except Alt–FdU (∼85% of control). In contrast, the effects of Zn2+ were highly sequence dependent. The effects of Zn2+ on the control sequence were similar to the effects of Mg2+ with a moderate concentration-dependent decrease in EtBr fluorescence. The effect of Zn2+ on the ‘Alt–FdU’ sequence was also moderate. In contrast, Zn2+ had a dramatic effect on EtBr fluorescence for the 3′-FdU and 5′-FdU sequences. Addition of 1 mM Zn2+ resulted in attenuation of EtBr fluorescence to <25% of control for these two sequences (Figure 3). The results are consistent with Zn2+ forming specific complexes with DNA sequences containing consecutive FdU nucleotides. Similar complexes do not form with Mg2+ or with sequences containing non-consecutive FdU nucleotides.Figure 3.

Bottom Line: Mg(2+) neither inhibited EtBr complexation nor had as strong of a stabilizing effect.DNA sequences that did not contain consecutive FdU were not stabilized by Zn(2+).A lipofectamine preparation of the Zn(2+)-DNA complex displayed enhanced cytotoxicity toward prostate cancer cells relative to the individual components prepared as lipofectamine complexes indicating the potential utility of Zn(2+)-DNA complexes for cancer treatment.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Biology, Program in Molecular Genetics, Wake Forest University School of Medicine, Winston-Salem, NC 27157, USA.

ABSTRACT
We report, based on semi-empirical calculations, that Zn(2+) binds duplex DNA containing consecutive FdU-dA base pairs in the major groove with distorted trigonal bipyramidal geometry. In this previously uncharacterized binding motif, O4 and F5 on consecutive FdU are axial ligands while three water molecules complete the coordination sphere. NMR spectroscopy confirmed Zn(2+) complexation occurred with maintenance of base pairing while a slight hypsochromic shift in circular dichroism (CD) spectra indicated moderate structural distortion relative to B-form DNA. Zn(2+) complexation inhibited ethidium bromide (EtBr) intercalation and stabilized FdU-substituted duplex DNA (ΔT(m) > 15 °C). Mg(2+) neither inhibited EtBr complexation nor had as strong of a stabilizing effect. DNA sequences that did not contain consecutive FdU were not stabilized by Zn(2+). A lipofectamine preparation of the Zn(2+)-DNA complex displayed enhanced cytotoxicity toward prostate cancer cells relative to the individual components prepared as lipofectamine complexes indicating the potential utility of Zn(2+)-DNA complexes for cancer treatment.

Show MeSH
Related in: MedlinePlus