Extended Data Fig. 4: Sso2081 and Sso1393 cA4-degradation mechanism investigated by TLC.
From: Ring nucleases deactivate type III CRISPR ribonucleases by degrading cyclic oligoadenylate
Csm-generated cOA (lane 1) was incubated with 2 μM Sso2081 dimer at 60 °C to determine the intermediate (Y) and final (X) reaction product over time (lanes 2–10). Lanes 11–13 show reaction product seen in lanes 2, 4 and 6, 5′-end phosphorylated using T4 PNK for identification of reaction intermediates and products by comparison to the 5′-end phosphorylated HO-A2>P and HO-A4>P standards generated with MazF nuclease. Lane 14 and lane 15 show the reaction products of 2 μM Sso1393 dimer incubated with cA4 at 70 °C for 20 and 120 min, respectively. Reaction product from lanes 14 and 15 are 5′-end phosphorylated by PNK for comparison with P-A2>P and P-A4>P standards. Comparison of PNK-treated reaction product to standards showed the presence of a low amount of intermediate (P-Y) during the Sso2081 cA4-cleavage reaction, which migrated similarly to the P-A4>P standard and did not change in abundance over time, whereas the abundance of the final product (P-X) increased over time. By contrast, comparison of Sso1393 PNK-treated reaction products at 20 min and 120 min showed a decrease of the intermediate (P-Y) over time and an increase of product (P-X).
