Intramolecularly folded G-quadruplex and i-motif structures in the proximal promoter of the vascular endothelial growth factor gene.
A polyguanine/polycytosine (polyG/polyC) tract in the proximal promoter of the vascular endothelial growth factor (VEGF) gene is essential for transcriptional activation. The guanine-rich (G-rich) and cytosine-rich (C-rich) strands on this tract are shown to form specific secondary structures, characterized as G-quadruplexes and i-motifs, respectively. Mutational analysis of the G-rich strand combined with dimethyl sulfate (DMS) footprinting, a polymerase stop assay, and circular dichroism (CD) spectroscopy revealed that the G-quadruplex containing a 1:4:1 double-chain reversal loop is the most thermodynamically stable conformation that this strand readily adopts. These studies provide strong evidence that the size of loop regions plays a critical role in determining the most favored folding pattern of a G-quadruplex. The secondary structure formed on the complementary C-rich strand was also determined by mutational analysis combined with Br(2) footprinting and CD spectroscopy. Our results reveal that at a pH of 5.9 this strand is able to form an intramolecular i-motif structure that involves six C-C(+) base pairs and a 2:3:2 loop configuration. Taken together, our results demonstrate that the G-quadruplex and i-motif structures are able to form on the G- and C-rich strands, respectively, of the polyG/polyC tract in the VEGF proximal promoter under conditions that favor the transition from B-DNA to non-B-DNA conformations.
Guo
Kexiao
K
Department of Biochemistry and Molecular Biophysics, University of Arizona, Tucson, AZ 85721, USA.
Gokhale
Vijay
V
Hurley
Laurence H
LH
Sun
Daekyu
D
D002942:Circular Dichroism; D003596:Cytosine / Q000737:chemistry; D004247:DNA / Q000737:chemistry; D018983:DNA Footprinting; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D054856:G-Quadruplexes; D006801:Humans; D007536:Isomerism; D009690:Nucleic Acid Conformation; D011401:Promoter Regions, Genetic; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics*
D042461:Vascular Endothelial Growth Factor A; D003596:Cytosine; D004247:DNA; D004259:DNA-Directed DNA Polymerase