DDX43 prefers single strand substrate and its full binding activity requires physical connection of all domains

Abstract 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.