DNA-PK autophosphorylation facilitates Artemis endonuclease activity

Goodarzi, Aaron A, Yu, Yaping, Riballo, Enriqueta, Douglas, Pauline, Walker, Sarah A, Ye, Ruiqiong, Harer, Christine, Marchetti, Caterina, Morrice, Nick, Jeggo, Penny A and Lees-Miller, Susan P (2006) DNA-PK autophosphorylation facilitates Artemis endonuclease activity. EMBO Journal, 25 (16). pp. 3880-3889. ISSN 0261-4189

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The 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.

Item Type: Article
Additional Information: 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.
Schools and Departments: School of Life Sciences > Sussex Centre for Genome Damage and Stability
Depositing User: Aaron Goodarzi
Date Deposited: 06 Feb 2012 18:23
Last Modified: 14 Jun 2012 08:32
URI: http://srodev.sussex.ac.uk/id/eprint/16038
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