Biophysical investigation of G-quadruplex recognition by the N-terminal construct of RNA helicase associated with AU-rich element (RHAU)
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Date
2013-12-06
Authors
Marushchak, Oksana
Journal Title
Journal ISSN
Volume Title
Publisher
American Society for Biochemistry and Molecular Biology (United States)
Abstract
G-quadruplexes, characterized by stacked G-tetrad rings held together by Hoogsteen hydrogen bonds, have been visualized in human cells and implicated in transcriptional and translational control, telomere maintenance and disease. RHA Helicase associated with AU-rich element (RHAU), a DEAH-box helicase, is a major G-quadruplex resolvase in human cell lysates. It binds G-quadruplexes through the RHAU specific motif in its N-terminus. In order to investigate the recognition of G-quadruplexes by helicases, the binding between the N-terminal construct of RHAU, RHAU53-105, and the DNA analog of the quadruplex formed by the 5’ terminus of human telomerase RNA component, hTR1-20, was investigated in a comprehensive biophysical approach followed by crystallization screening. RHAU53-105, hTR1-20 DNA and their complexes were analysed by gel electrophoresis, UV-visible spectroscopy, spectropolarimetry, dynamic light scattering and small angle X-ray scattering (SAXS). The findings reveal that hTR1-20 DNA, separated in two conformations by size exclusion chromatography in the presence of potassium cations, assumes a disk-like parallel G-quadruplex secondary structure in solution. Far-UV circular dichroism spectra and SAXS demonstrate that RHAU53-105 assumes an extended (Dmax = 7.8 nm , rG = 2.1 (±0.2) nm) and ordered conformation in solution. The analysis confirms the binding between RHAU53-105 and each conformation of the hTR1-20 DNA quadruplex. Circular dichroism spectra indicate the retention of quadruplex secondary structure in both RHAU53-105•hTR1-20 DNAc1 and RHAU53-105•hTR1-20 DNAc2 complexes. This analysis provides some insight into the interaction between G-quadruplexes and the N-terminal domain of RHAU and identifies 0.2 M sodium formate, 20 % (w/v) polyethylene glycol 3350 and 1.5 M sodium chloride, 10 % (v/v) ethanol as preliminary conditions for crystallization of the complex of RHAU53-105 and hTR1-20 DNAc2.
Description
Keywords
G-quadruplex, RNA Helicase, Human telomerase reverse transcriptase RNA, RNA Helicase Associated with AU-rich Element, G41R, DHX36, hTR
Citation
Meier, M., Patel, T. R., Booy, E. P., Marushchak, O., Okun, N., Deo, S., Howard, R., McEleney, K., Harding, S. E., Stetefeld, J., and McKenna, S. a. (2013) Binding of G-quadruplexes to the N-terminal recognition domain of the RNA helicase associated with AU-rich element (RHAU). J. Biol. Chem. 288, 35014–27.