A conserved quadruplex motif located in a transcription activation site of the human c-kit oncogene.
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.
Fernando
Himesh
H
Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW, United Kingdom.
Reszka
Anthony P
AP
Huppert
Julian
J
Ladame
Sylvain
S
Rankin
Sarah
S
Venkitaraman
Ashok R
AR
Neidle
Stephen
S
Balasubramanian
Shankar
S
D020816:Amino Acid Motifs; D000818:Animals; D001483:Base Sequence; D002942:Circular Dichroism; D004247:DNA; D054856:G-Quadruplexes; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D009690:Nucleic Acid Conformation; D009857:Oncogenes; D002679:Pan troglodytes; D019009:Proto-Oncogene Proteins c-kit / Q000235:genetics*; D051381:Rats; D013056:Spectrophotometry, Ultraviolet; D015533:Transcriptional Activation / Q000235:genetics*
D004247:DNA; D019009:Proto-Oncogene Proteins c-kit