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The Dicer-related helicases (DRHs) are members of a helicase subfamily, and mammalian DRHs such as retinoic acid-inducible gene-I (RIG-I), are involved in antiviral immunity. Caenorhabditis elegans DRH-1 and DRH-3 play crucial roles in antiviral function and chromosome segregation, respectively. Although intrinsic double-stranded RNA-dependent ATP-hydrolyzing activity has been observed in the recombinant DRH-3 protein prepared from Escherichia coli, there are no reports of biochemical studies of the nematode RIG-I homolog DRH-1. In this study, the secondary structure prediction by JPred4 revealed that DRH-1 and DRH-3 had distinct N-terminal regions and that a 200-amino acid N-terminal region of DRH-1 could form a structure very rich in α-helices. We investigated expressions and purifications of a codon-optimized DRH-1 with four different N-terminal tags, identifying poly-histidine (His)-small ubiquitin-like modifier (SUMO) as a suitable tag for DRH-1 preparation. Full-length (isoform a) and a N-terminal truncated (isoform b) of DRH-1 were purified as the His-SUMO-tagged fusion proteins. Finally, the nucleic acid-dependent ATPase activities were investigated for the two His-SUMO-tagged DRH-1 isoforms and His-tagged DRH-3. The tagged DRH-3 exhibited dsRNA-dependent ATPase activity. However, detectable dsRNA dependency of ATPase activities was not found in either isoform of tagged DRH-1 and a tag-free DRH-1 (isoform a) treated with SUMO protease. These observations suggest that DRH-1 and its short isoform have no or poor nucleic acid-dependent ATPase activity, unlike DRH-3 and mammalian DRHs. [ABSTRACT FROM AUTHOR]

The yeast Dbp9p is a member of the DEAD box family of RNA helicases, which are thought to be involved in RNA metabolism. Dbp9p seems to function in ribosomal RNA biogenesis, but it has not been biochemically characterized. To analyze the enzymatic characteristics of the protein, we expressed a recombinant Dbp9p in Escherichia coli and purified it to homogeneity. The purified protein exhibited RNA unwinding and binding activity in the absence of NTP, and this activity was abolished by a mutation in the RNA-binding domain. We then characterized the ATPase activity of Dbp9p with respect to cofactor specificity; the activity was found to be severely inhibited by yeast total RNA and moderately inhibited by poly(U), poly(A), and poly(C) but to be stimulated by yeast genomic DNA and salmon sperm DNA. In addition, Dbp9p exhibited DNA-DNA and DNA-RNA helicase activity in the presence of ATP. These results indicate that Dbp9p has biochemical characteristics unique among DEAD box proteins. [ABSTRACT FROM AUTHOR]