Biochemical Characterization of DDX43 (HAGE) Helicase
Talwar, Tanu 1988-
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DDX43, DEAD-box polypeptide 43, also known as HAGE (helicase antigen gene), is a member of the DEAD-box family of RNA helicases. It is highly expressed in many tumor types compared with normal tissues, and is therefore considered as a potential target for immunotherapy of cancers. Despite its unique expression and potential as a therapy target, little is known about its biochemical and physiological functions. In this study, we purified recombinant DDX43 protein to near homogeneity and our gel filtration results showed that DDX43 exists as a monomer in solution. Biochemical assays using monomer fractions of DDX43 demonstrated that it could unwind both RNA and DNA substrates in an ATP-dependent manner, and most efficiently in the presence of Mg2+; no significant unwinding activity was detected with other nucleoside triphosphates or divalent cations. Replacing the conserved lysine in motif I (K292A) or aspartic acid in motif II (D396A) abolished the unwinding activity. Intriguingly, DDX43 could unwind RNA substrates without strict polarity, but it showed higher unwinding activity on a 5’ tail RNA substrate compared to a 3’ tail or blunt end RNA substrates. However, for DNA substrates, it exhibited unidirectional translocation and unwound DNA in a 3’to 5’ direction only. A K-homology (KH) domain in the N-terminal region of DDX43 was found to possess strong nucleic acid binding ability and the N-terminal domain showed novel strand exchange and required for the unwinding activity. Compared to the full-length protein, the C-terminal helicase domain had weaker unwinding activity, but an increase was observed in the presence of the N-terminal domain. Using Co-immunoprecipitation and mass spectrometry, we found that DDX43 associates with pICln and MEP50, two methylosome subunits that are involved in assembly of the spliceosome complex. Collectively, our results suggest that DDX43 is a KH domain containing ATP-dependent dual helicase, where unwinding activity is mediated through cooperation between its N-terminal domain and helicase domain, and potentially involved in pre-mRNA splicing.
DegreeMaster of Science (M.Sc.)
CommitteeLee, Jeremy; Moore, Stanley; Lukong, Erique K; Kobryn, Kerry
Copyright DateMarch 2017
DEAD-Box, Helicase, KH-Domain