Substrate Specificities of DDX1: A Human DEAD-Box Protein

DDX1 is a human DEAD-box RNA helicase involved in various stages of RNA metabolism, from transcription to decay, and is consequently implicated in many human diseases. The nucleotides hydrolyzed by DDX1 and the structures of the nucleic acids upon which it acts in cells remain largely unknown. In this study, we identify the nucleic acid sequences and structures that support DDX1's nucleotide hydrolysis activity and determine its nucleotide hydrolysis specificity. Our data demonstrate that DDX1 hydrolyzes only ATP and deoxy-ATP in the presence of RNA. The ATP hydrolysis activity of DDX1 is stimulated by single-stranded RNA molecules as short as ten nucleotides, a blunt-ended double-stranded RNA, double-stranded RNA/DNA hybrid, and single-stranded DNA. Under our experimental conditions, single-stranded DNA stimulates DDX1's ATPase activity to a smaller extent compared to the other RNA constructs or the RNA/DNA hybrid. Given DDX1's involvement in numerous critical cellular processes and its implication in various human diseases, determining its substrate specificity not only enhances our understanding of its in vivo function, but also facilitates the development of novel therapeutic approaches.