Limits...
Kinetics and mechanism of G-quadruplex formation and conformational switch in a G-quadruplex of PS2.M induced by Pb²⁺.

Liu W, Zhu H, Zheng B, Cheng S, Fu Y, Li W, Lau TC, Liang H - Nucleic Acids Res. (2012)

Bottom Line: UV-melting curves demonstrate that the Pb(2+)-induced G-quadruplex formed unimolecularly and the highest melting temperature (T(m)) is 72°C.Kinetic studies suggest that the Pb(2+)-induced folding of PS2.M to G-quadruplex probably proceeds through a three-step pathway involving two intermediates.Comparison of the relaxation times shows that the Na(+)→Pb(2+) exchange is more facile than the K(+)→Pb(2+) exchange process, and the mechanisms for these processes are proposed.

View Article: PubMed Central - PubMed

Affiliation: CAS Key Laboratory of Soft Matter Chemistry, Hefei National Laboratory for Physical Sciences at Microscale, University of Science and Technology of China, Hefei, Anhui 230026, P R China.

ABSTRACT
DNA sequences with guanine repeats can form G-quartets that adopt G-quadruplex structures in the presence of specific metal ions. Using circular dichroism (CD) and ultraviolet-visible (UV-Vis) spectroscopy, we determined the spectral characteristics and the overall conformation of a G-quadruplex of PS2.M with an oligonucleotide sequence, d(GTG(3)TAG(3)CG(3)TTG(2)). UV-melting curves demonstrate that the Pb(2+)-induced G-quadruplex formed unimolecularly and the highest melting temperature (T(m)) is 72°C. The analysis of the UV titration results reveals that the binding stoichiometry of Pb(2+) ions to PS2.M is two, suggesting that the Pb(2+) ions coordinate between adjacent G-quartets. Binding of ions to G-rich DNA is a complex multiple-pathway process, which is strongly affected by the type of the cations. Kinetic studies suggest that the Pb(2+)-induced folding of PS2.M to G-quadruplex probably proceeds through a three-step pathway involving two intermediates. Structural transition occurs after adding Pb(NO(3))(2) to the Na(+)- or K(+)-induced G-quadruplexes, which may be attributed to the replacement of Na(+) or K(+) by Pb(2+) ions and the generation of a more compact Pb(2+)-PS2.M structure. Comparison of the relaxation times shows that the Na(+)→Pb(2+) exchange is more facile than the K(+)→Pb(2+) exchange process, and the mechanisms for these processes are proposed.

Show MeSH

Related in: MedlinePlus

Spectrophotometric titration of PS2.M (2.5 µM) with of Pb(NO3)2. (a) Spectral changes in the titration process. The final cation concentrations after mixing are shown at the right of the panel; (b) Plot of absorbance change at 303 nm versus the mole ratio of Pb(NO3)2 to PS2.M. The temperature was maintained at 25°C.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3351173&req=5

gkr1310-F2: Spectrophotometric titration of PS2.M (2.5 µM) with of Pb(NO3)2. (a) Spectral changes in the titration process. The final cation concentrations after mixing are shown at the right of the panel; (b) Plot of absorbance change at 303 nm versus the mole ratio of Pb(NO3)2 to PS2.M. The temperature was maintained at 25°C.

Mentions: Previous studies have shown that the UV absorption spectrum of the Pb2+-driven folded G-quadruplex possesses an absorption maximum at ∼303 nm, which is distinctly different from that of the unfolded oligonucleotide (15,23). To monitor the expected absorbance change and cation binding number, we titrated PS2.M with Pb(NO3)2 at micromolar concentrations at 25°C. The absorbance changes at 303 nm were monitored as a function of the molar ratio of the cation to the oligomer and the results are shown in Figure 2. The difference spectrum was obtained by subtracting the absorption spectrum from the spectrum of the fully unfolded oligomer. As indicated in Figure 2a, the absorbance at 303 nm increases with increasing concentration of Pb(NO3)2, which is accompanied by relatively smaller absorbance decrease in absorbance at ∼245 and ∼275 nm. Figure 2b shows the plot of absorbance change at 303 nm versus the [Pb2+]/[PS2.M] molar ratio. The result is consistent with the binding stoichiometry of two Pb2+ ions per oligonucleotide, which can coordinate a Pb2+ ion between each of the three quartets. This is contrast to a previous suggestion that only one Pb2+ ion binds to one oligonucleotide (29). The ability of Pb2+ ions to stabilize G-quadruplexes at micromolar concentrations indicates the high affinity of the Pb2+ ions for the DNA. Moreover, no spectral changes were observed changes when KNO3 was added to the G-quadruplex of PS2.M induced by Pb2+ ions, while spectral changes occurred when Pb(NO3)2 was added to the G-quadruplex induced by K+ ions, again indicating the high stability of Pb2+–PS2.M complex. Based on these observations, the genotoxicity of Pb2+ ion may arise partially from its strong binding to a quadruplex formation in the genome; more closely, in the promoter regions where the quadruplexes can serve as transcription switches. Thus, if Pb2+ can displace K+ from the quadruplex, but not vice versa, the switching function may be hindered, providing an explanation for genotoxicity.Figure 2.


Kinetics and mechanism of G-quadruplex formation and conformational switch in a G-quadruplex of PS2.M induced by Pb²⁺.

Liu W, Zhu H, Zheng B, Cheng S, Fu Y, Li W, Lau TC, Liang H - Nucleic Acids Res. (2012)

Spectrophotometric titration of PS2.M (2.5 µM) with of Pb(NO3)2. (a) Spectral changes in the titration process. The final cation concentrations after mixing are shown at the right of the panel; (b) Plot of absorbance change at 303 nm versus the mole ratio of Pb(NO3)2 to PS2.M. The temperature was maintained at 25°C.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3351173&req=5

gkr1310-F2: Spectrophotometric titration of PS2.M (2.5 µM) with of Pb(NO3)2. (a) Spectral changes in the titration process. The final cation concentrations after mixing are shown at the right of the panel; (b) Plot of absorbance change at 303 nm versus the mole ratio of Pb(NO3)2 to PS2.M. The temperature was maintained at 25°C.
Mentions: Previous studies have shown that the UV absorption spectrum of the Pb2+-driven folded G-quadruplex possesses an absorption maximum at ∼303 nm, which is distinctly different from that of the unfolded oligonucleotide (15,23). To monitor the expected absorbance change and cation binding number, we titrated PS2.M with Pb(NO3)2 at micromolar concentrations at 25°C. The absorbance changes at 303 nm were monitored as a function of the molar ratio of the cation to the oligomer and the results are shown in Figure 2. The difference spectrum was obtained by subtracting the absorption spectrum from the spectrum of the fully unfolded oligomer. As indicated in Figure 2a, the absorbance at 303 nm increases with increasing concentration of Pb(NO3)2, which is accompanied by relatively smaller absorbance decrease in absorbance at ∼245 and ∼275 nm. Figure 2b shows the plot of absorbance change at 303 nm versus the [Pb2+]/[PS2.M] molar ratio. The result is consistent with the binding stoichiometry of two Pb2+ ions per oligonucleotide, which can coordinate a Pb2+ ion between each of the three quartets. This is contrast to a previous suggestion that only one Pb2+ ion binds to one oligonucleotide (29). The ability of Pb2+ ions to stabilize G-quadruplexes at micromolar concentrations indicates the high affinity of the Pb2+ ions for the DNA. Moreover, no spectral changes were observed changes when KNO3 was added to the G-quadruplex of PS2.M induced by Pb2+ ions, while spectral changes occurred when Pb(NO3)2 was added to the G-quadruplex induced by K+ ions, again indicating the high stability of Pb2+–PS2.M complex. Based on these observations, the genotoxicity of Pb2+ ion may arise partially from its strong binding to a quadruplex formation in the genome; more closely, in the promoter regions where the quadruplexes can serve as transcription switches. Thus, if Pb2+ can displace K+ from the quadruplex, but not vice versa, the switching function may be hindered, providing an explanation for genotoxicity.Figure 2.

Bottom Line: UV-melting curves demonstrate that the Pb(2+)-induced G-quadruplex formed unimolecularly and the highest melting temperature (T(m)) is 72°C.Kinetic studies suggest that the Pb(2+)-induced folding of PS2.M to G-quadruplex probably proceeds through a three-step pathway involving two intermediates.Comparison of the relaxation times shows that the Na(+)→Pb(2+) exchange is more facile than the K(+)→Pb(2+) exchange process, and the mechanisms for these processes are proposed.

View Article: PubMed Central - PubMed

Affiliation: CAS Key Laboratory of Soft Matter Chemistry, Hefei National Laboratory for Physical Sciences at Microscale, University of Science and Technology of China, Hefei, Anhui 230026, P R China.

ABSTRACT
DNA sequences with guanine repeats can form G-quartets that adopt G-quadruplex structures in the presence of specific metal ions. Using circular dichroism (CD) and ultraviolet-visible (UV-Vis) spectroscopy, we determined the spectral characteristics and the overall conformation of a G-quadruplex of PS2.M with an oligonucleotide sequence, d(GTG(3)TAG(3)CG(3)TTG(2)). UV-melting curves demonstrate that the Pb(2+)-induced G-quadruplex formed unimolecularly and the highest melting temperature (T(m)) is 72°C. The analysis of the UV titration results reveals that the binding stoichiometry of Pb(2+) ions to PS2.M is two, suggesting that the Pb(2+) ions coordinate between adjacent G-quartets. Binding of ions to G-rich DNA is a complex multiple-pathway process, which is strongly affected by the type of the cations. Kinetic studies suggest that the Pb(2+)-induced folding of PS2.M to G-quadruplex probably proceeds through a three-step pathway involving two intermediates. Structural transition occurs after adding Pb(NO(3))(2) to the Na(+)- or K(+)-induced G-quadruplexes, which may be attributed to the replacement of Na(+) or K(+) by Pb(2+) ions and the generation of a more compact Pb(2+)-PS2.M structure. Comparison of the relaxation times shows that the Na(+)→Pb(2+) exchange is more facile than the K(+)→Pb(2+) exchange process, and the mechanisms for these processes are proposed.

Show MeSH
Related in: MedlinePlus