Formation of a unique end-to-end stacked pair of G-quadruplexes in the hTERT core promoter with implications for inhibition of telomerase by G-quadruplex-interactive ligands.
The hTERT core promoter contains a G-rich region of 12 consecutive G-tracts, embracing 3 Sp1 binding sites, and has the potential to form multiple G-quadruplexes. From the 12 runs of guanines, 9 putative hTERT G-quadruplex-forming sequences were selected to assay for G-quadruplex formation and stability using circular dichroism and a Taq polymerase stop assay. Results from biophysical and chemical assays demonstrate an approximate inverse correlation between total loop size and structure stability. Investigation of the full-length hTERT G-rich sequence using a Taq polymerase stop assay and dimethyl sulfate footprinting revealed the formation of a unique end-to-end stacked G-quadruplex structure from this sequence. This structure consists of an all parallel G-quadruplex, formed by four consecutive G-tracts, linked to another, atypical G-quadruplex, formed by two pairs of consecutive G-tracts separated by a 26-base loop. This 26-base loop likely forms a stable hairpin structure, which would explain the unexpected stability of this G-quadruplex. Significantly, the formation of this tandem G-quadruplex structure in the full-length sequence masks all three Sp1 binding sites, which is predicted to produce significant inhibition of hTERT promoter activity. Furthermore, our study implies that inhibition of telomerase activity by some G-quadruplex ligands is not only produced by targeting telomeric G-quadruplexes but also by stabilization of the hTERT promoter G-quadruplexes.
Palumbo
SunMi L
SL
Arizona Cancer Center, University of Arizona, 1515 North Campbell Avenue, Tucson, Arizona 85724, USA.
Ebbinghaus
Scot W
SW
Hurley
Laurence H
LH
D001483:Base Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D054856:G-Quadruplexes; D006801:Humans; D008024:Ligands; D009690:Nucleic Acid Conformation; D011401:Promoter Regions, Genetic; D016329:Sp1 Transcription Factor / Q000378:metabolism; D019098:Telomerase / Q000235:genetics*
D008024:Ligands; D016329:Sp1 Transcription Factor; D019098:Telomerase