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DNA-PK autophosphorylation facilitates Artemis endonuclease activity
journal contribution
posted on 2023-06-07, 19:47 authored by Aaron A Goodarzi, Yaping Yu, Enriqueta Riballo, Pauline Douglas, Sarah A Walker, Ruiqiong Ye, Christine Harer, Caterina Marchetti, Nick Morrice, Penny Jeggo, Susan P Lees-MillerThe Artemis nuclease is defective in radiosensitive severe combined immunodeficiency patients and is required for the repair of a subset of ionising radiation induced DNA double-strand breaks (DSBs) in an ATM and DNA-PK dependent process. Here, we show that Artemis phosphorylation by ATM and DNA-PK in vitro is primarily attributable to S503, S516 and S645 and demonstrate ATM dependent phosphorylation at serine 645 in vivo. However, analysis of multisite phosphorylation mutants of Artemis demonstrates that Artemis phosphorylation is dispensable for endonuclease activity in vitro and for DSB repair and V(D)J recombination in vivo. Importantly, DNA-dependent protein kinase catalytic subunit (DNA-PKcs) autophosphorylation at the T2609-T2647 cluster, in the presence of Ku and target DNA, is required for Artemis-mediated endonuclease activity. Moreover, autophosphorylated DNA-PKcs stably associates with Ku-bound DNA with large single-stranded overhangs until overhang cleavage by Artemis. We propose that autophosphorylation triggers conformational changes in DNA-PK that enhance Artemis cleavage at single-strand to double-strand DNA junctions. These findings demonstrate that DNA-PK autophosphorylation regulates Artemis access to DNA ends, providing insight into the mechanism of Artemis mediated DNA end processing.
History
Publication status
- Published
Journal
EMBO JournalISSN
0261-4189Publisher
Nature Publishing GroupExternal DOI
Issue
16Volume
25Page range
3880-3889Department affiliated with
- Sussex Centre for Genome Damage Stability Publications
Notes
Jeggo Penny: Designed project. 80% of work done in PJ lab, co-wrote paper. Disproved exisitng model and showed that Artemis is activated by DNA-PK auto-phosphorylation.Full text available
- No
Peer reviewed?
- Yes
Legacy Posted Date
2012-02-06Usage metrics
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