title: ATM and DNA-PK function redundantly to phosphorylate H2AX after exposure to ionizing radiation.
abstract: H2AX phosphorylation is an early step in the response to DNA damage. It is widely accepted that ATM (ataxia telangiectasia mutated protein) phosphorylates H2AX in response to DNA double-strand breaks (DSBs). Whether DNA-dependent protein kinase (DNA-PK) plays any role in this response is unclear. Here, we show that H2AX phosphorylation after exposure to ionizing radiation (IR) occurs to similar extents in human fibroblasts and in mouse embryo fibroblasts lacking either DNA-PK or ATM but is ablated in ATM-deficient cells treated with LY294002, a drug that specifically inhibits DNA-PK. Additionally, we show that inactivation of both DNA-PK and ATM is required to ablate IR-induced H2AX phosphorylation in chicken cells. We confirm that H2AX phosphorylation induced by DSBs in nonreplicating cells is ATR (ataxia telangiectasia and Rad3-related protein) independent. Taken together, we conclude that under most normal growth conditions, IR-induced H2AX phosphorylation can be carried out by ATM and DNA-PK in a redundant, overlapping manner. In contrast, DNA-PK cannot phosphorylate other proteins involved in the checkpoint response, including chromatin-associated Rad17. However, by phosphorylating H2AX, DNA-PK can contribute to the presence of the damage response proteins MDC1 and 53BP1 at the site of the DSB.
authors.affiliation: Genome Damage and Stability Centre, University of Sussex, East Sussex, United Kingdom.
authors.forename: Tom
authors.initials: T
authors.lastname: Stiff
authors.forename: Mark
authors.initials: M
authors.lastname: O'Driscoll
authors.forename: Nicole
authors.initials: N
authors.lastname: Rief
authors.forename: Kuniyoshi
authors.initials: K
authors.lastname: Iwabuchi
authors.forename: Markus
authors.initials: M
authors.lastname: Löbrich
authors.forename: Penny A
authors.initials: PA
authors.lastname: Jeggo
mesh_terms: D000818:Animals; D001260:Ataxia Telangiectasia / Q000378:metabolism / Q000473:pathology; D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins / Q000378:metabolism / Q000528:radiation effects; D002461:Cell Line, Transformed; D002645:Chickens; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D006657:Histones / Q000378:metabolism*; D006801:Humans; D007259:Infrared Rays; D009687:Nuclear Proteins; D010766:Phosphorylation / Q000187:drug effects / Q000528:radiation effects; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000528:radiation effects; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D025521:Tumor Suppressor Proteins
chemical_list: D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; C468783:H2AX protein, human; D006657:Histones; D009687:Nuclear Proteins; C113575:Rad17 protein, human; D016159:Tumor Suppressor Protein p53; D025521:Tumor Suppressor Proteins; C576897:Atr protein, mouse; C576324:ATM protein, human; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C576325:Atm protein, mouse; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases