Characterisation of novel ATM-interacting factors involved in double-strand break repair
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Ataxia-telangiectasia (A-T) is a rare human disease characterized by cerebellar degeneration, immune system defects and cancer predisposition. The disease has been the subject of intense scientific scrutiny, particularly since the identification in 1995 of the gene mutated in A-T, ATM (Savitzky et al). The ataxia-telangiectasia mutated protein (ATM) has emerged as a central player in the cellular response to ionizing radiation (IR), in which it plays a critical, although incompletely characterised, role in the activation of cell cycle checkpoints that lead to DNA damage-induced arrest at G1/S, S and G2/M. The ATM protein is a member of the phosphatidylinositol 3-kinase-like family of ¿atypical¿ serine/threonine protein kinases (PIKKs). Other members of this protein family include ATM- and Rad3-related (ATR), DNA-dependent protein kinase catalytic subunit (DNA-PKcs), mammalian target of rapamycin (mTOR), and ATX/hSMG-1. The main aim of this study is to utilize the DT40 model system to elucidate the regulation and function of Atm. We have used the efficient gene targeting possible in this cell line to modify the Atm locus to produce a fully functional, but epitope tagged Atm protein. Using the novel TAP tag we have generated we have purified TAP-tagged Atm protein. In parallel, similar approaches have allowed us to purify in parallel similarly TAP tagged Atr and 53BP1. SILAC-assisted mass spectrometry has allowed us to identify a large set of novel putative 'interactors', including many proteins involved in either binding or remodelling chromatin. Selected 'interactors' from this screen have been confirmed in co-immunoprecipitation experiments using extracts prepared from human cells. Our latest results from a selected subset of these confirmed ATM interacting proteins will be presented.
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