A Conserved Quadruplex Motif Located in a Transcription Activation Site of the Human c-kit Oncogene

• Published in: Biochemistry (Easton) Vol. 45; no. 25; pp. 7854 - 7860
• Main Authors: Fernando, Himesh, Reszka, Anthony P, Huppert, Julian, Ladame, Sylvain, Rankin, Sarah, Venkitaraman, Ashok R, Neidle, Stephen, Balasubramanian, Shankar
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 27.06.2006
• Subjects: Amino Acid Motifs; Animals; Base Sequence; Circular Dichroism; DNA; G-Quadruplexes; Humans; Mice; Molecular Sequence Data; Nuclear Magnetic Resonance, Biomolecular; Nucleic Acid Conformation; Oncogenes; Pan troglodytes; Proto-Oncogene Proteins c-kit - genetics; Rats; Spectrophotometry, Ultraviolet; Transcriptional Activation - genetics
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi0601510

The c-kit gene encodes a receptor tyrosine kinase, whose engagement by its ligand triggers signals leading to cell proliferation. c-kit activity is elevated in gastrointestinal stromal tumors (GISTs), and its therapeutic inhibition by small molecules such as imatinib is clinically validated. We identified a putative quadruplex forming 21-nucleotide sequence upstream of the c-kit transcription initiation site (c-kit21), on the G-rich strand, which occupies a site required for core promoter activity. Here, we show by nuclear magnetic resonance (NMR), circular dichroism (CD), and ultraviolet (UV) spectroscopic methods that c-kit21 forms quadruplexes under physiological conditions. Mutational analysis of c-kit21 has provided insights into its structural polymorphism. In particular, one mutated form appears to form a single quadruplex species that adopts a parallel conformation. The quadruplex-forming sequence shows a high level of sequence conservation across human, mouse, rat, and chimpanzee. The small variation in sequence between the quadruplex in human/chimpanzee as compared to the rat/mouse was examined more closely by biophysical methods. Despite a variation in the sequence and length of loop 2, the quadruplexes showed both comparable CD spectra, indicative of parallel quadruplexes, and also similar thermal-stability profiles, suggesting conservation of biophysical characteristics. Collectively, the evidence suggests that this quadruplex is a serious target for a detailed functional investigation at the cell-biology level.