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• Due to its cancer-specific expression, DDX43 (DEAD-box helicase 43) is considered a potential target molecule for cancer therapy. • Biochemical assays to determine the enzymatic activities and properties of DDX43 protein, including its KH domain, link domain, and ATPase/helicase core domain. • Identify the specific nucleic acids sequence bound by its KH domain using SELEX, ChIP-seq, CLIP-seq, and NMR. • Structural characterization of the KH domain and full-length DDX43 protein aided by Phyre 2, NMR, and AphaFold. • Emerging roles of DDX43 in piRNA amplification, tumorigenesis, RAS signaling, and innate immunity. • Structural and functional studies of DDX43 will facilitate anti-cancer drug development by targeting DDX43. DDX43 (DEAD-box helicase 43), also known as HAGE (helicase antigen gene), is a member of the DEAD-box protein family. It contains a K homology (KH) domain in its N terminus, a helicase core domain in its C terminus, and a flexible linker domain in between. DDX43 expression is low or undetectable in normal tissue, but is overexpressed in many tumors; therefore, it is considered a potential target molecule for cancer therapy. We, along with other groups, have shown that DDX43 is an ATP-dependent RNA and DNA helicase, and the KH domain is required for its ATPase and unwinding activity. Electrophoretic mobility shift assay (EMSA), SELEX (systematic evolution of ligands by exponential enrichment), chromatin immunoprecipitation (ChIP)-seq, crosslinking immunoprecipitation (CLIP)-seq, and nuclear magnetic resonance (NMR) showed that the KH domain prefers to bind pyrimidine-rich ssDNA and ssRNA, such as TTGT in the promoter regions of genes. Moreover, the KH domain facilitates the substrate specificity and processivity of the DDX43 helicase. No animal model has been generated for DDX43; cellular studies have revealed that DDX43 has roles in piRNA amplification, tumorigenesis, RAS signaling, and innate immunity. Structural and functional studies of DDX43 will not only advance our understanding of DEAD-box helicases and KH domains, but also shed light on the application of DDX43 as therapeutics, where its key binding sites can be targeted by small molecules and natural products as an alternative approach in treating DDX43 overexpressed cancers. [ABSTRACT FROM AUTHOR]

DDX43 is a cancer/testis antigen and is thought to stimulate oncogenic pathways in cell proliferation, while its specific function in cancer development is largely unexplored. DDX43 is the member of RNA helicase in DEAD-box family, consists of conserved helicase core and a single K-homology (KH) domain in its N-terminus. In this paper, we expressed and purified human DDX43 protein in E. coli and demonstrated that this protein is a homogeneous monomer. To understand the role and explore the substrates preference of DDX43 in vitro , we systematically studied its binding properties. We found that DDX43 prefers single-strand DNA or RNA with length longer than 12 nt and much prefers guanosine than the other three nucleotides. Achievement of the full binding affinity of protein to substrate needs the existence of all domains, and they must be connected. The absence of either of them or the disjunction can result in a decreased binding affinity to substrates, approximately reduced 10-fold. We also found that the unwinding ability of DDX43 in vitro was neither efficient nor sustainable. • DDX43 prefers single strand G-rich substrate. • Both KH domain and helicase core are required in substrates binding. • KH domain and helicase core must be linked for DDX43 high affinity to substrates. [ABSTRACT FROM AUTHOR]