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. Author manuscript; available in PMC: 2024 Mar 7.
Published in final edited form as: Nature. 2020 Feb 26;579(7798):291–296. doi: 10.1038/s41586-020-2041-2

Fig. 4 |. U3 snoRNA drives assembly, activation, and auto-phosphorylation of DNA-PK at the T2609 cluster.

Fig. 4 |

a, DNA-PKcs and KU86 RT stops that map to the four pre-rRNA introns; 5′ETS, ITS1, ITS2, and 3′ETS. irCLIP was performed in biological duplicate. b, Transcript normalized histogram of DNA-PKcs (orange) and KU86 (blue) irCLIP RT stops from DMSO-treated HeLa cells mapping to U3. c, In silico (mFold)-predicted secondary structure of U3. The top three and two peaks of DNA-PKcs and KU86, respectively, are annotated on the secondary structure. The length of the 5′end stem-loop is shown. d, Baculovirus-purified human DNA-PK in vitro kinase phosphorylation assay in the presence of increasing amounts of U3-SL1. Western blots were performed with antibodies against DNA-PKcs phosphorylated at the T2609 cluster (top), total DNA-PK (middle), and KU86 (bottom). e, Baculovirus-purified human DNA-PK in vitro kinase phosphorylation assay against purified human TP53. Activated DNA-PK phosphorylates TP53 on serine 15. An in vitro ATM kinase assay was used as a control. Western blots were performed with antibodies against TP53 phosphorylated on serine 15 (top), total TP53 (middle) and KU86 (bottom). d, e, n = 3 biologically independent experiments.