Penicud, K; (2012) The role of ATM regulatory proteins in DNA damage signalling. Doctoral thesis , UCL (University College London).

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Abstract

The DNA damage activated checkpoint kinase ATM is mutated in ataxia telangiectasia, which is characterised by cancer predisposition and neural degeneration. After ionising radiation (IR) ATM signalling proceeds via interaction with Nijmegen breakage syndrome 1 (NBS1). Conversely, ATM INteractor (ATMIN) is required for ATM activation after hypotonic shock. Full ATM activation also requires concomitant chromatin modifications, including histone H4 acetylation at K16 (H4K16Ac) by Tip60. However, the molecular mechanisms underpinning ATM pathway choice and physiological stimuli for ATMIN dependent ATM signalling were unclear. The aims of my PhD studies were to (a) establish physiological roles for ATMIN dependent ATM signalling, (b) investigate the mechanisms and consequences of ATM co-factor choice, and (c) identify novel ATM regulatory proteins. I characterised ATMIN’s role in preventing DNA damage accumulation in the embryo. Moreover, ATMIN suppresses DNA damage at the telomere. Loss of ATMIN from telomerase deficient cells exacerbates telomere dysfunction. Conversely, ATMIN is dispensable for oncogene induced senescence after colonic KRasG12D expression. To investigate ATM pathway choice I generated nbs1∆/∆; atmin∆/∆ MEFs. These compound mutants exhibit reduced senescence and increased proliferation compared to nbs1∆/∆, despite increased endogenous DNA damage. A similar phenotype was observed in the intestine. Both nbs1∆/∆;atmin∆/∆ MEFs and intestinally-deleted nbs1∆G;atmin∆G mice were extremely radiosensitive. Furthermore, ATMIN deletion augments IR-induced ATM substrate phosphorylation. Thus, competition between ATMIN and NBS1 for ATM interaction is fundamental to ATM signalling and disrupting this balance is deleterious to cells. An shRNA screen identified DMAP1 as a novel ATM regulator. DMAP1 deficient cells are radiosensitive, and exhibit impaired ATM signalling after both IR and hypotonic shock. DMAP1 interacts with Tip60, and is required for Tip60 acetylation of H4K16 after DNA damage. Histone deacetylase inhibitors rescued ATM substrate phosphorylation and H4K16Ac levels after IR in DMAP1 depleted cells. Thus DMAP1 is a novel regulator of ATM function.

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