Extension of G-quadruplex DNA by ciliate telomerase.
Telomeric DNA can fold into four-stranded structures known as G-quadruplexes. Here we investigate the ability of G-quadruplex DNA to serve as a substrate for recombinant Tetrahymena and native Euplotes telomerase. Inter- and intramolecular G-quadruplexes were gel-purified and their stability examined using native gel electrophoresis, circular dichroism (CD) and thermal denaturation. While intermolecular G-quadruplexes were highly stable, they were excellent substrates for both ciliate telomerases in primer extension assays. In contrast, intramolecular G-quadruplexes formed in K+ exhibited biphasic unfolding and were not extended by ciliate telomerases. Na+-stabilised intramolecular G-quadruplexes were extended by telomerase owing to their rapid rate of dissociation. The Tetrahymena telomerase protein component bound to inter- but not intramolecular K+-stabilised G-quadruplexes. This study provides evidence that parallel intermolecular G-quadruplexes can serve as substrates for telomerase in vitro, their extension being mediated through direct interactions between this higher-order structure and telomerase.
Oganesian
Liana
L
Children's Medical Research Institute, Sydney, Australia.
Moon
Ian K
IK
Bryan
Tracy M
TM
Jarstfer
Michael B
MB
D000818:Animals; D002942:Circular Dichroism; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D016812:Euplotes / Q000201:enzymology*; D054856:G-Quadruplexes; D006358:Hot Temperature; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D011188:Potassium / Q000494:pharmacology; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012964:Sodium / Q000494:pharmacology; D013379:Substrate Specificity; D019098:Telomerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013768:Tetrahymena / Q000201:enzymology*
D011994:Recombinant Proteins; D004247:DNA; D012964:Sodium; D019098:Telomerase; D011188:Potassium