Biochemical Characterization of G4 Quadruplex Telomerase RNA Unwinding by the RNA Helicase RHAU
G4 quadruplexes are stable secondary structures prevalent in DNA and RNA that exhibit diverse regulatory functions. Herein, we describe an in vitro technique using the purified RNA helicase RHAU to unwind a G4 quadruplex identified near the 5′ end of the human telomerase RNA (hTR). A synthetic RNA corresponding to the quadruplex forming region of hTR (hTR10–43), as well as a predicted complementary strand (25P1), are combined in a reaction containing the purified helicase and ATP. Reaction products and appropriate controls are resolved by native gel electrophoresis. Gels can be stained using a combination of total RNA and quadruplex-specific dyes to observe the expected quadruplex to duplex conversion. This straightforward method can be extended to study structural changes in other inter- or intramolecular quadruplex containing DNA/RNA molecules with the RHAU helicase or other RNA/DNA remodeling enzymes.
Key wordsRHAU Quadruplex Helicase Telomerase RNA DHX36 G4R1 P1-helix Unwinding Secondary structure
Evan Booy is supported by the Manitoba Health Research Council postdoctoral fellowship. This work is supported by the Canadian Institutes of Health Research (CIHR)/Manitoba Health Research Council (MHRC) regional partnership program and a Cancer Research Society Operating Grant.
- 23.Fu JJ, Li LY, Lu GX (2002) Molecular cloning and characterization of human DDX36 and mouse Ddx36 genes, new members of the DEAD/H box superfamily. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai). Acta biochimica et biophysica Sinica 34:655–661Google Scholar
- 24.Creacy SD, Routh ED, Iwamoto F et al (2008) G4 resolvase 1 binds both DNA and RNA tetramolecular quadruplex with high affinity and is the major source of tetramolecular quadruplex G4-DNA and G4-RNA resolving activity in HeLa cell lysates. J Biol Chem 283:34626–34634PubMedCentralPubMedCrossRefGoogle Scholar