PMC

NSP13 possesses ATP dependent dsRNA unwinding activity. A. Recombinant SARS-CoV-2 NSP13 protein purification. SDS-PAGE analysis of the purified NSP13 protein, and the gel was stained with Coomassie Blue. B. Western blot analysis of the purified NSP13 protein with anti-Myc antibody. C&D. ATPase activity of NSP13. (C) The ATPase activities of wild type (WT) and mutant (D374A/E375A) SARS-CoV-2 NSP13, the data were calculated by the Michaelis–Menten equation. (D) The Kcat, Km and Kcat/Km values of NSP13 WT and D374A/E375A mutant. E. Schematic illustration of the FRET-based dsRNA unwinding assay. Two complementary nucleic acid strands are labeled with Cy3 and the quencher BHQ2, respectively. The fluorescence of Cy3 is quenched by BHQ2 upon annealing of the RNA. With the unwinding of RNA duplexes by NSP13 protein, the fluorescence of Cy3 increases. F. The unwinding activity of SARS-CoV-2 NSP13 in the presence or absence of ATP. G. The unwinding assay of WT and mutant (D374A/E375A) NSP13.H&I. The unwinding rate increases with the increment of NSP13 concentration (0, 10, 20 and 50 nM). (H) Real-time dsRNA unwinding kinetics. (I) Initial unwinding rate of NSP13 at different concentrations. The initial unwinding rate was calculated by the kinetics in the first 5min. J. The unwinding activity of NSP13 upon dsRNA with different length of duplexes region (20, 25, 35, 50 and 60 bp). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

The rigid connection between stalk and helicase is essential to helicase activity but not ATPase activity. A&B. Overall structures of (A) Upf1 (PDB ID: 2wjy) and (B) SARS-CoV-2 NSP13 (PDB ID:6zsl) which were colored by different domains. The stalk domain was colored in magenta. C. Superimposition of NSP13 stalk domains from SARS-CoV-2 (magenta) and MERS-CoV (blue, PDB ID:5wwp). D. Schematic illustration of WT NSP13 and linker insertion mutants (Z-linker-SH and ZS-linker-H). E. Western blot analysis of the purified WT NSP13 and linker insertion mutants with anti-Myc antibody. F&G. Linker insertion did not affect ATPase activity of NSP13. (F) ATPase activity analysis of WT NSP13 and linker insertion mutants. (G) The Kcat, Km and Kcat/Km values of WT NSP13 and linker insertion mutants. H&I. Linker insertion between stalk and helicase domains abolished dsRNA unwinding activity of NSP13. (H) The unwinding assay of WT NSP13 and linker insertion mutants. (I) Initial unwinding rate of WT NSP13 and linker insertion mutants. The initial unwinding rate was calculated by kinetics in the first 5min. The significance was analyzed by t-test, ∗∗∗p < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Duplex unwinding activities of DpTV Hel and its mutants. (A–D) Purified MBP-Hel was incubated with standard dsRNA, dsDNA or RNA/DNA hybrid substrates in the presence or absence of Mg²⁺ or ATP (RNA: green line; DNA: blue line; HEX: red asterisk). The preparations of unwinding substrates are presented in the “Materials and methods” section. Substrates were incubated in standard reaction mixtures (except as noted) and the unwinding activity was assessed by gel electrophoresis followed by scan with a Typhoon9200. (A) Standard dsRNA substrates. (B) D^⁎/R substrates. (C) R^⁎/D substrates. (D) D^⁎/D substrates. Lanes 1 and 2: reaction mixtures lacking enzyme that were either left native (lane 1) or boiled (lane 2); Lane 3, complete reaction mixture with negative control MBP alone; lanes 4 and 5, complete reaction mixtures with MgCl₂ omitted (lane 4) or with ATP omitted (lane 5); lane 6, complete reaction mixture. (E) dsRNA unwinding activities of DpTV Hel mutants. Lanes 1 and 2: reaction mixtures lacking enzyme that were either left native (lane 1) or boiled (lane 2); lanes 3, 4, 5 and 6, complete reaction mixture with MBP-Hel (lane 3), MBP-Hel (I) (lane 4), MBP-Hel (II) (lane 5) or MBP-Hel (VI) (lane 6). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Structure and domains of Bos taurus Dhx36 in complex with the parallel quadruplex from the MYC promoter (PDB 5VHE). The DSM domain (dark red) is primarily responsible for the G4 (orange) recognition by contacting the exposed G4-tetrad through its hydrophobic surface. The interaction is complemented by the OB domain (purple) and RecA1 (light blue) engaging the G4 backbone by creating a positively-charge “cage”, which also contributes to the proposed unwinding mechanism. The N-terminal linker (NL) is depicted in white. Rotations refer to the upper-left model. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)