title: DNA-PK autophosphorylation facilitates Artemis endonuclease activity.
abstract: 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.
authors.affiliation: Genome Damage and Stability Centre, University of Sussex, East Sussex, UK.
authors.forename: Aaron A
authors.initials: AA
authors.lastname: Goodarzi
authors.forename: Yaping
authors.initials: Y
authors.lastname: Yu
authors.forename: Enriqueta
authors.initials: E
authors.lastname: Riballo
authors.forename: Pauline
authors.initials: P
authors.lastname: Douglas
authors.forename: Sarah A
authors.initials: SA
authors.lastname: Walker
authors.forename: Ruiqiong
authors.initials: R
authors.lastname: Ye
authors.forename: Christine
authors.initials: C
authors.lastname: Härer
authors.forename: Caterina
authors.initials: C
authors.lastname: Marchetti
authors.forename: Nick
authors.initials: N
authors.lastname: Morrice
authors.forename: Penny A
authors.initials: PA
authors.lastname: Jeggo
authors.forename: Susan P
authors.initials: SP
authors.lastname: Lees-Miller
mesh_terms: D020134:Catalytic Domain; D004247:DNA / Q000737:chemistry; D004265:DNA Helicases / Q000737:chemistry; D004260:DNA Repair; D004277:DNA, Single-Stranded / Q000737:chemistry; D051747:DNA-Activated Protein Kinase / Q000737:chemistry*; D004268:DNA-Binding Proteins; D004720:Endonucleases / Q000737:chemistry / Q000235:genetics; D006801:Humans; D000072200:Ku Autoantigen; D009687:Nuclear Proteins / Q000737:chemistry* / Q000235:genetics; D010766:Phosphorylation; D011487:Protein Conformation; D011995:Recombination, Genetic; D012694:Serine / Q000737:chemistry
chemical_list: D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D012694:Serine; D004247:DNA; D051747:DNA-Activated Protein Kinase; C426344:DCLRE1C protein, human; D004720:Endonucleases; D004265:DNA Helicases; C106893:XRCC5 protein, human; D000072200:Ku Autoantigen