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Solution structures of all parallel-stranded monomeric and dimeric G-quadruplex scaffolds of the human c-kit2 promoter.

Kuryavyi V, Phan AT, Patel DJ - Nucleic Acids Res. (2010)

Bottom Line: These NMR-based studies are now extended to the c-kit2 promoter, which adopts two distinct all-parallel-stranded conformations in slow exchange, one of which forms a monomeric G-quadruplex (form-I) in 20 mM K(+)-containing solution and the other a novel dimeric G-quadruplex (form-II) in 100 mM K(+)-containing solution.The c-kit2 promoter dimeric form-II G-quadruplex adopts an unprecedented all-parallel-stranded topology where individual c-kit2 promoter strands span a pair of three-G-tetrad-layer-containing all-parallel-stranded G-quadruplexes aligned in a 3' to 5'-end orientation, with stacking continuity between G-quadruplexes mediated by a sandwiched A•A non-canonical pair.We propose that strand exchange during recombination events within guanine-rich segments, could potentially be mediated by a synapsis intermediate involving an intergenic parallel-stranded dimeric G-quadruplex.

View Article: PubMed Central - PubMed

Affiliation: Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA.

ABSTRACT
Previous studies have demonstrated that nuclease hypersensitivity regions of several proto-oncogenic DNA promoters, situated upstream of transcription start sites, contain guanine-rich tracts that form intramolecular G-quadruplexes stabilized by stacked G•G•G•G tetrads in monovalent cation solution. The human c-kit oncogenic promoter, an important target in the treatment of gastrointestinal tumors, contains two such stretches of guanine-rich tracts, designated c-kit1 and c-kit2. Our previous nuclear magnetic resonance (NMR)-based studies reported on the novel G-quadruplex scaffold of the c-kit1 promoter in K(+)-containing solution, where we showed for the first time that even an isolated guanine was involved in G-tetrad formation. These NMR-based studies are now extended to the c-kit2 promoter, which adopts two distinct all-parallel-stranded conformations in slow exchange, one of which forms a monomeric G-quadruplex (form-I) in 20 mM K(+)-containing solution and the other a novel dimeric G-quadruplex (form-II) in 100 mM K(+)-containing solution. The c-kit2 promoter dimeric form-II G-quadruplex adopts an unprecedented all-parallel-stranded topology where individual c-kit2 promoter strands span a pair of three-G-tetrad-layer-containing all-parallel-stranded G-quadruplexes aligned in a 3' to 5'-end orientation, with stacking continuity between G-quadruplexes mediated by a sandwiched A•A non-canonical pair. We propose that strand exchange during recombination events within guanine-rich segments, could potentially be mediated by a synapsis intermediate involving an intergenic parallel-stranded dimeric G-quadruplex.

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Imino proton NMR spectral patterns associated with form-I and form-II of the c-kit2 promoter. (a) Chemical sequence of the c-kit2 21-mer sequence. The guanine residues involved in G-quadruplex formation are shown in bold. (b) Imino proton NMR spectrum of a 1:1 mixture of c-kit2 T21 promoter form-I and form-II in slow equilibrium in 20 mM KCl, 5 mM phosphate, H2O buffer, pH 6.8 at 43°C. (c) Imino proton NMR spectrum of the c-kit2 T12/T21 promoter form-I in 20 mM KCl, 5 mM phosphate, H2O buffer, pH 6.8, at 25°C, with assignments listed over the spectrum. (d) Imino proton NMR spectrum of the c-kit2 T21 promoter form-II in 100 mM KCl, 5 mM phosphate, H2O buffer, pH 6.8, at 25°C, with assignments listed over the spectrum. The protocols for sample preparation of spectra in (b) to (d) are descried in text (e) Non-denaturing polyacrylamide gel electrophoresis (PAGE) analysis of c-kit2 T12/T21 (form-I) and c-kit2 T21 (form-II) in K+-containing solution. Migration markers are provided: 93del, an interlocked dimeric G-quadruplex (Phan et al., 2005); J19: a stacked dimeric G-quadruplex (Phan,A.T., unpublished data); J19 2T1: a monomeric propeller-type G-quadruplex (Phan,A.T., unpublished data).
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Figure 1: Imino proton NMR spectral patterns associated with form-I and form-II of the c-kit2 promoter. (a) Chemical sequence of the c-kit2 21-mer sequence. The guanine residues involved in G-quadruplex formation are shown in bold. (b) Imino proton NMR spectrum of a 1:1 mixture of c-kit2 T21 promoter form-I and form-II in slow equilibrium in 20 mM KCl, 5 mM phosphate, H2O buffer, pH 6.8 at 43°C. (c) Imino proton NMR spectrum of the c-kit2 T12/T21 promoter form-I in 20 mM KCl, 5 mM phosphate, H2O buffer, pH 6.8, at 25°C, with assignments listed over the spectrum. (d) Imino proton NMR spectrum of the c-kit2 T21 promoter form-II in 100 mM KCl, 5 mM phosphate, H2O buffer, pH 6.8, at 25°C, with assignments listed over the spectrum. The protocols for sample preparation of spectra in (b) to (d) are descried in text (e) Non-denaturing polyacrylamide gel electrophoresis (PAGE) analysis of c-kit2 T12/T21 (form-I) and c-kit2 T21 (form-II) in K+-containing solution. Migration markers are provided: 93del, an interlocked dimeric G-quadruplex (Phan et al., 2005); J19: a stacked dimeric G-quadruplex (Phan,A.T., unpublished data); J19 2T1: a monomeric propeller-type G-quadruplex (Phan,A.T., unpublished data).

Mentions: Samples shown on Figures 1b and 5a–c were prepared by method of gel-filtration through Sephadex G-25 packed in spin-down columns. The remaining samples were prepared by equilibrium dialysis (24,28,29). The strand concentration of the samples varied from 0.2 to 4.0 mM (measured using millimolar extinction coefficients 202 and 204 for forms I and II, respectively) and the solution was either 20 mM KCl, 5 mM K-phosphate buffer, pH 6.8 (form-I) or 100 mM KCl, 5 mM K-phosphate buffer, pH 6.8 (form-II).Figure 1.


Solution structures of all parallel-stranded monomeric and dimeric G-quadruplex scaffolds of the human c-kit2 promoter.

Kuryavyi V, Phan AT, Patel DJ - Nucleic Acids Res. (2010)

Imino proton NMR spectral patterns associated with form-I and form-II of the c-kit2 promoter. (a) Chemical sequence of the c-kit2 21-mer sequence. The guanine residues involved in G-quadruplex formation are shown in bold. (b) Imino proton NMR spectrum of a 1:1 mixture of c-kit2 T21 promoter form-I and form-II in slow equilibrium in 20 mM KCl, 5 mM phosphate, H2O buffer, pH 6.8 at 43°C. (c) Imino proton NMR spectrum of the c-kit2 T12/T21 promoter form-I in 20 mM KCl, 5 mM phosphate, H2O buffer, pH 6.8, at 25°C, with assignments listed over the spectrum. (d) Imino proton NMR spectrum of the c-kit2 T21 promoter form-II in 100 mM KCl, 5 mM phosphate, H2O buffer, pH 6.8, at 25°C, with assignments listed over the spectrum. The protocols for sample preparation of spectra in (b) to (d) are descried in text (e) Non-denaturing polyacrylamide gel electrophoresis (PAGE) analysis of c-kit2 T12/T21 (form-I) and c-kit2 T21 (form-II) in K+-containing solution. Migration markers are provided: 93del, an interlocked dimeric G-quadruplex (Phan et al., 2005); J19: a stacked dimeric G-quadruplex (Phan,A.T., unpublished data); J19 2T1: a monomeric propeller-type G-quadruplex (Phan,A.T., unpublished data).
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Figure 1: Imino proton NMR spectral patterns associated with form-I and form-II of the c-kit2 promoter. (a) Chemical sequence of the c-kit2 21-mer sequence. The guanine residues involved in G-quadruplex formation are shown in bold. (b) Imino proton NMR spectrum of a 1:1 mixture of c-kit2 T21 promoter form-I and form-II in slow equilibrium in 20 mM KCl, 5 mM phosphate, H2O buffer, pH 6.8 at 43°C. (c) Imino proton NMR spectrum of the c-kit2 T12/T21 promoter form-I in 20 mM KCl, 5 mM phosphate, H2O buffer, pH 6.8, at 25°C, with assignments listed over the spectrum. (d) Imino proton NMR spectrum of the c-kit2 T21 promoter form-II in 100 mM KCl, 5 mM phosphate, H2O buffer, pH 6.8, at 25°C, with assignments listed over the spectrum. The protocols for sample preparation of spectra in (b) to (d) are descried in text (e) Non-denaturing polyacrylamide gel electrophoresis (PAGE) analysis of c-kit2 T12/T21 (form-I) and c-kit2 T21 (form-II) in K+-containing solution. Migration markers are provided: 93del, an interlocked dimeric G-quadruplex (Phan et al., 2005); J19: a stacked dimeric G-quadruplex (Phan,A.T., unpublished data); J19 2T1: a monomeric propeller-type G-quadruplex (Phan,A.T., unpublished data).
Mentions: Samples shown on Figures 1b and 5a–c were prepared by method of gel-filtration through Sephadex G-25 packed in spin-down columns. The remaining samples were prepared by equilibrium dialysis (24,28,29). The strand concentration of the samples varied from 0.2 to 4.0 mM (measured using millimolar extinction coefficients 202 and 204 for forms I and II, respectively) and the solution was either 20 mM KCl, 5 mM K-phosphate buffer, pH 6.8 (form-I) or 100 mM KCl, 5 mM K-phosphate buffer, pH 6.8 (form-II).Figure 1.

Bottom Line: These NMR-based studies are now extended to the c-kit2 promoter, which adopts two distinct all-parallel-stranded conformations in slow exchange, one of which forms a monomeric G-quadruplex (form-I) in 20 mM K(+)-containing solution and the other a novel dimeric G-quadruplex (form-II) in 100 mM K(+)-containing solution.The c-kit2 promoter dimeric form-II G-quadruplex adopts an unprecedented all-parallel-stranded topology where individual c-kit2 promoter strands span a pair of three-G-tetrad-layer-containing all-parallel-stranded G-quadruplexes aligned in a 3' to 5'-end orientation, with stacking continuity between G-quadruplexes mediated by a sandwiched A•A non-canonical pair.We propose that strand exchange during recombination events within guanine-rich segments, could potentially be mediated by a synapsis intermediate involving an intergenic parallel-stranded dimeric G-quadruplex.

View Article: PubMed Central - PubMed

Affiliation: Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA.

ABSTRACT
Previous studies have demonstrated that nuclease hypersensitivity regions of several proto-oncogenic DNA promoters, situated upstream of transcription start sites, contain guanine-rich tracts that form intramolecular G-quadruplexes stabilized by stacked G•G•G•G tetrads in monovalent cation solution. The human c-kit oncogenic promoter, an important target in the treatment of gastrointestinal tumors, contains two such stretches of guanine-rich tracts, designated c-kit1 and c-kit2. Our previous nuclear magnetic resonance (NMR)-based studies reported on the novel G-quadruplex scaffold of the c-kit1 promoter in K(+)-containing solution, where we showed for the first time that even an isolated guanine was involved in G-tetrad formation. These NMR-based studies are now extended to the c-kit2 promoter, which adopts two distinct all-parallel-stranded conformations in slow exchange, one of which forms a monomeric G-quadruplex (form-I) in 20 mM K(+)-containing solution and the other a novel dimeric G-quadruplex (form-II) in 100 mM K(+)-containing solution. The c-kit2 promoter dimeric form-II G-quadruplex adopts an unprecedented all-parallel-stranded topology where individual c-kit2 promoter strands span a pair of three-G-tetrad-layer-containing all-parallel-stranded G-quadruplexes aligned in a 3' to 5'-end orientation, with stacking continuity between G-quadruplexes mediated by a sandwiched A•A non-canonical pair. We propose that strand exchange during recombination events within guanine-rich segments, could potentially be mediated by a synapsis intermediate involving an intergenic parallel-stranded dimeric G-quadruplex.

Show MeSH
Related in: MedlinePlus