Propeller-type parallel-stranded G-quadruplexes in the human c-myc promoter - PubMed (original) (raw)

Propeller-type parallel-stranded G-quadruplexes in the human c-myc promoter

Anh Tuân Phan et al. J Am Chem Soc. 2004.

Abstract

The nuclease-hypersensitivity element III1 in the c-myc promoter is a good anticancer target since it largely controls transcriptional activation of the important c-myc oncogene. Recently, the guanine-rich strand of this element has been shown to form an equilibrium between G-quadruplex structures built from two different sets of G-stretches; two models of intramolecular fold-back antiparallel-stranded G-quadruplexes, called "basket" and "chair" forms, were proposed. Here, we show by NMR that two sequences containing these two sets of G-stretches form intramolecular propeller-type parallel-stranded G-quadruplexes in K(+)-containing solution. The two structures involve a core of three stacked G-tetrads formed by four parallel G-stretches with all anti guanines and three double-chain-reversal loops bridging three G-tetrad layers. The central loop contains two or six residues, while the two other loops contain only one residue.

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Figures

Figure 1

Figure 1

DNA sequences of the NHE G-rich strand from the c-myc promoter and its derivatives: (a) Pu27, the 27-nt wild-type sequence; (b) myc-2345, a “chair” derivative containing G-tracks number 2, 3, 4, and 5; and (c) myc-1245, a “basket” derivative containing G-tracks number 1, 2, 4, and 5; the G-track number 3 is substituted by T4.

Figure 2

Figure 2

One-dimensional 600 MHz proton spectra of (a) Pu27, (b) myc-2345, and (c) myc-1245. Experimental conditions: strand concentration, 1 mM; temperature, 25 °C; 70 mM KCl; potassium phosphate, 20 mM; pH 7.

Figure 3

Figure 3

Determination of stoichiometry by NMR titration of the equilibrium strand concentrations of the structured form and of the unfolded monomer. Squares and triangles represent myc-2345 and myc-1245, respectively. Lines with a slope of one are drawn through the data points. Experimental conditions: 7 mM KCl; potassium phosphate, 2 mM; pH 7; temperature, 60 °C for myc-2345 and 50 °C for myc-1245.

Figure 4

Figure 4

Imino proton NMR spectra of (a) myc-2345 and (b) myc-1245, with assignments listed over the reference spectrum (ref) at the top of the figure. Imino protons were assigned in 15N-filtered spectra of samples that were 2% (a) 15N-labeled and (b) 15N,13C-labeled at the indicated positions. The reference spectrum was recorded using the same pulse sequence but with a different phase cycle. Experimental conditions: 70 mM KCl; potassium phosphate, 20 mM; pH 7; temperature, 25 °C; strand concentration, 0.5–1 mM.

Figure 5

Figure 5

Through-bond correlations between imino and H8 protons via 13C5 at natural abundance for (b) myc-2345 and (c) myc-1245, using long-range J-couplings shown in (a). Assignments of guanine H8 protons, labeled with residue numbers, were obtained from the already-assigned imino protons. A peak from T(H6) is labeled with a star. Experimental conditions: 70 mM KCl; potassium phosphate, 20 mM; pH 7; temperature, 25 °C; strand concentration, 2 mM.

Figure 6

Figure 6

NOESY spectra (mixing time, 200 ms) of (a) myc-2345 and (a′) myc-1245 (same conditions as in Figure 5). The imino–H8 cross-peaks are framed and labeled with the number of imino protons in the first position and that of H8 in the second position. (b) Specific imino–H8 connectivity pattern around a G-tetrad (G_α_•G_β_•G_γ_•G_δ_) indicated with arrows (connectivity between G_δ_ and G_α_ implied). (c, c′) Schematic structures of myc-2345 and myc-1245 that satisfy NOE connectivities shown in parentheses.

Figure 7

Figure 7

Imino proton spectra of (a) myc-2345 and (b) myc-1245 in H2O (upper) and after 1 h in D2O (lower) showing peaks from the central G-tetrad. (c) Intensities (in arbitrary units, A.U.) of G21 imino protons in myc-2345 and myc-1245 as a function of time. Squares and triangles represent myc-2345 and myc-1245, respectively. Experimental conditions: strand concentration, 1 mM; temperature, 25 °C; 70 mM KCl; potassium phosphate, 20 mM; pH 7.

References

    1. Gartel AL, Shchors K. Exp Cell Res. 2003;283:17–21. - PubMed
    2. Pelengaris S, Khan M. Arch Biochem Biophys. 2003;416:129–136. - PubMed
    1. Slamon DJ, deKernion JB, Verma IM, Cline MJ. Science. 1984;224:256–262. - PubMed
    2. Marcu KB, Bossone SA, Patel AJ. Annu Rev Biochem. 1992;61:809–860. - PubMed
    3. Pelengaris S, Rudolph B, Littlewood T. Curr Opin Genet Dev. 2000;10:100–105. - PubMed
    1. Siebenlist U, Hennighausen L, Battey J, Leder P. Cell. 1984;37:381–391. - PubMed
    2. Cooney M, Czernuszewicz G, Postel EH, Flint SJ, Hogan ME. Science. 1988;241:456–459. - PubMed
    3. Davis TL, Firulli AB, Kinniburgh AJ. Proc Natl Acad Sci USA. 1989;86:9682–9686. - PMC - PubMed
    4. Berberich SJ, Postel EH. Oncogene. 1995;10:2343–2347. - PubMed
    1. Simonsson T, Pecinka P, Kubista M. Nucleic Acids Res. 1998;26:1167–1172. - PMC - PubMed
    1. Siddiqui-Jain A, Grand CL, Bearss DJ, Hurley LH. Proc Natl Acad Sci USA. 2002;99:11593–11598. - PMC - PubMed

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