Purification and Characterization of Double-Stranded Nucleic Acid-Dependent ATPase Activities of Tagged Dicer-Related Helicase 1 and its Short Isoform in Caenorhabditis elegans

• Published in: Genes 2020-07, Vol.11 (7), p.734
• Main Authors: Kobayashi, Taishi, Murakami, Takuro, Hirose, Yuu, Eki, Toshihiko
• Format: Article
• Language: English
• Subjects: Adenosine triphosphatase; Amino acids; Animals; Antibiotics; Caenorhabditis elegans; Caenorhabditis elegans Proteins - chemistry; Caenorhabditis elegans Proteins - genetics; Caenorhabditis elegans Proteins - metabolism; Catalytic Domain; Cloning; DEAD-box RNA Helicases - chemistry; DEAD-box RNA Helicases - genetics; DEAD-box RNA Helicases - metabolism; Deoxyribonucleic acid; DNA; DNA helicase; DNA polymerase; Double-stranded RNA; E coli; Genes; Histidine; Isoenzymes - chemistry; Isoenzymes - genetics; Isoenzymes - metabolism; Isoforms; Laboratories; Mammals; Mutagenesis; Nematodes; Plasmids; Polymerase chain reaction; Protein Conformation, alpha-Helical; Protein expression; Protein structure; Proteins; Retinoic acid; RNA, Double-Stranded - metabolism; Secondary structure; Signal transduction; Small Ubiquitin-Related Modifier Proteins - genetics; Small Ubiquitin-Related Modifier Proteins - metabolism; Ubiquitin
• ISSN: 2073-4425; 2073-4425
• DOI: 10.3390/genes11070734

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. 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 , 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.