Biophysical investigation of G-quadruplex recognition by the N-terminal construct of RNA helicase associated with AU-rich element (RHAU)
| dc.contributor.author | Marushchak, Oksana | |
| dc.contributor.examiningcommittee | McKenna, Sean (Chemistry) Court, Deborah (Microbiology) | en_US |
| dc.contributor.supervisor | Stetefeld, Jorg (Chemistry) | en_US |
| dc.date.accessioned | 2014-09-03T20:44:07Z | |
| dc.date.available | 2014-09-03T20:44:07Z | |
| dc.date.issued | 2013-12-06 | en_US |
| dc.degree.discipline | Chemistry | en_US |
| dc.degree.level | Master of Science (M.Sc.) | en_US |
| dc.description.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. | en_US |
| dc.description.note | October 2014 | en_US |
| dc.identifier.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. | en_US |
| dc.identifier.uri | http://hdl.handle.net/1993/23958 | |
| dc.language.iso | eng | en_US |
| dc.publisher | American Society for Biochemistry and Molecular Biology (United States) | en_US |
| dc.rights | open access | en_US |
| dc.subject | G-quadruplex | en_US |
| dc.subject | RNA Helicase | en_US |
| dc.subject | Human telomerase reverse transcriptase RNA | en_US |
| dc.subject | RNA Helicase Associated with AU-rich Element | en_US |
| dc.subject | G41R | en_US |
| dc.subject | DHX36 | en_US |
| dc.subject | hTR | en_US |
| dc.title | Biophysical investigation of G-quadruplex recognition by the N-terminal construct of RNA helicase associated with AU-rich element (RHAU) | en_US |
| dc.type | master thesis | en_US |