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DNA‐PK autophosphorylation facilitates Artemis endonuclease activity
Author(s) -
Goodarzi Aaron A,
Yu Yaping,
Riballo Enriqueta,
Douglas Pauline,
Walker Sarah A,
Ye Ruiqiong,
Härer Christine,
Marchetti Caterina,
Morrice Nick,
Jeggo Penny A,
LeesMiller Susan P
Publication year - 2006
Publication title -
the embo journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.484
H-Index - 392
eISSN - 1460-2075
pISSN - 0261-4189
DOI - 10.1038/sj.emboj.7601255
Subject(s) - biology , autophosphorylation , endonuclease , dna , genetics , computational biology , microbiology and biotechnology , biochemistry , phosphorylation , protein kinase a
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.