Structural studies on RNA helicase associated with AU-Rich element (RHAU) and DExD-box helicase 21 (DDX21)

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Ghosh, Dhruba
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G-quadruplexes (G4s) are physiologically significant tetra-stranded thermodynamically stable structures, characterized by stacked G- quartets, formed by guanine stretches on a single or multiple DNA/RNA strands, and implicated in various diseases. Helicase proteins capable of unfolding G4 structures include RNA helicase associated with AU-rich element (RHAU) and DExD-box helicase 21 (DDX21), that unwind both RNA and DNA G4 structures. In order to perform biophysical studies of the RHAU protein, full length RHAU (RHAUFL) and a RHAU construct lacking its N-terminal 50 amino acid glycine-rich region (RHAU51-1008) were tested in the optimization of the expression of the RHAU proteins in E. coli. In this thesis, we purified the RHAUFL protein using a comprehensive recombinant protein production approach involving gel electrophoresis, Western blot, cobalt affinity chromatography, size exclusion chromatography (SEC) and anion exchange chromatography. DDX21 binds G4 through its C-terminal domain. In order to have insight into its structure and G4 recognition, C-terminal DDX21 protein construct DDX21C209 was expressed in E. coli, and purified using nickel affinity chromatography. Biophysical approaches including circular dichroism (CD) spectropolarimetry and isothermal titration calorimetry were used to check the quality of the DDX21C209 protein, a thermal shift assay was used to investigate thermal stability of DDX21C209, SEC and gel electrophoresis were performed following crystallization screening with and without a 22-nucleotide truncated human telomere DNA G4, hTel. In order to perform biophysical studies, full length DDX21 (DDX21FL) protein was expressed in E. coli and its purification was attempted using nickel affinity chromatography. Crystallization of the G4 helicase proteins both in a free state and in complex with G4 structures would lead to a better understanding of G4 helicase structure and function.
Structural Studies, Helicase, RNA Helicase, RHAU, DHX36, DDX21