Abstract
The K-homology (KH) domain is a nucleic acid-binding domain present in many proteins but has not been reported in helicases. DDX43, also known as HAGE (helicase antigen gene), is a member of the DEAD-box protein family. It contains a helicase core domain in its C terminus and a potential KH domain in its N terminus. DDX43 is highly expressed in many tumors and is, therefore, considered a potential target for immunotherapy. Despite its potential as a therapeutic target, little is known about its activities. Here, we purified recombinant DDX43 protein to near homogeneity and found that it exists as a monomer in solution. Biochemical assays demonstrated that it is an ATP-dependent RNA and DNA helicase. Although DDX43 was active on duplex RNA regardless of the orientation of the single-stranded RNA tail, it preferred a 5' to 3' polarity on RNA and a 3' to 5' direction on DNA. Truncation mutations and site-directed mutagenesis confirmed that the KH domain in DDX43 is responsible for nucleic acid binding. Compared with the activity of the full-length protein, the C-terminal helicase domain had no unwinding activity on RNA substrates and had significantly reduced unwinding activity on DNA. Moreover, the full-length DDX43 protein, with single amino acid change in the KH domain, had reduced unwinding and binding activates on RNA and DNA substrates. Our results demonstrate that DDX43 is a dual helicase and the KH domain is required for its full unwinding activity.
Highlights
The K-homology (KH) domain is a nucleic acid-binding domain present in many proteins but has not been reported in helicases
First we aimed to identify the best NTP for the unwinding activity of DDX43 and found that efficient unwinding was observed with ATP and dATP on the 5Ј-tailed 13-bp duplex RNA substrate (Fig. 4A), which is consistent with the previous findings for DEAD-box helicases, whereas both ATP and dATP were effective on the forked duplex DNA (Fig. 4B)
We discovered that the KH domain in the N-terminal region is involved in nucleic acid binding and is essential for the efficient unwinding activity of DDX43 helicase
Summary
The K-homology (KH) domain is a nucleic acid-binding domain present in many proteins but has not been reported in helicases. DDX43, known as HAGE (helicase antigen gene), is a member of the DEAD-box protein family It contains a helicase core domain in its C terminus and a potential KH domain in its N terminus. The full-length DDX43 protein, with single amino acid change in the KH domain, had reduced unwinding and binding activates on RNA and DNA substrates. Helicases are molecular motors that transduce the chemical energy generated by ATP hydrolysis into an oligonucleotide strand separation and protein displacement They are involved in virtually all aspects of nucleic acid metabolism, including replication, repair, recombination, transcription, chromosome segregation, and telomere maintenance [1,2,3]. DEAD-box helicases possess a nonprocessive separation activity, and most RNA helicases examined to date do not efficiently unwind duplexes with more than one-and-a-half-helical turns in vitro, but they efficiently separate shorter duplexes [2]. The authors declare that they have no conflicts of interest with the contents of this article
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