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. 2015 Mar 16;112(13):3949–3954. doi: 10.1073/pnas.1419409112

Fig. 3.

Fig. 3.

Roles of the individual domains of hOAS3 in sensing dsRNA. (A) Catalytic properties of individual domains of hOAS3 in the presence of dsRNA (2.5 OD260 poly I:C) and two divalent metal ions, Mg2+ or Mn2+. (B) Inactivation of hOAS3 by point mutations in the active site of domain DIII. Reactions contained full-length OAS3 (0.1 μM). (C) Active site mutagenesis in hOAS1 and hOAS3. Reactions contained 0.1 μM hOAS1 or hOAS3.DI, 2.5 OD260 poly I:C, 1 mM ATP and trace 32P-α-ATP, and were conducted at 37 °C for 2 h. Samples were resolved by 20% denaturing PAGE and visualized by phosphorimaging. (D, Left) activation of hOAS1 by dsRNA18 (Methods). (Right) Competition of hOAS3.DI and hOAS1 for binding to dsRNA18. WT hOAS3.DI (Ki = 3.74 μM) binds dsRNA stronger than hOAS1 (Kdapp = 50 μM), whereas two hOAS3.DI mutants that disrupt contacts to dsRNA (R30A and R41A) do not exhibit a detectable competition. Reactions contained 0.5 μM hOAS1 (Left), 1 μM hOAS1 (Right), and 100 nM dsRNA18 (Right). Data points show mean ± SD determined from at least two replicate time courses.