DNA-PK autophosphorylation facilitates Artemis endonuclease activity.
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.
Goodarzi
Aaron A
AA
Genome Damage and Stability Centre, University of Sussex, East Sussex, UK.
Yu
Yaping
Y
Riballo
Enriqueta
E
Douglas
Pauline
P
Walker
Sarah A
SA
Ye
Ruiqiong
R
Härer
Christine
C
Marchetti
Caterina
C
Morrice
Nick
N
Jeggo
Penny A
PA
Lees-Miller
Susan P
SP
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
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