Analysis of Chk2, ATM, NBS1, three proteins involved in the cellular response to DNA damage

Buscemi, Giacomo (2007). Analysis of Chk2, ATM, NBS1, three proteins involved in the cellular response to DNA damage. PhD thesis The Open University.



In presence of damage to nuclear DNA, eukaryotic cells trigger a set of signal transduction pathways, which lead to the arrest of cell-cycle progression until repair has occurred, to the increase of DNA repair capacity or, in some cases to a controlled suicide (apoptosis). In human cells in presence of DNA damage Chk2 kinase protein is activated by ATM, the protein mutated in ataxia telangiectasia (AT), and targets several substrates involved in the global DNA damage response.

I demonstrated that Chk2 activation after DNA damage induction by ɣ-radiation treatment requires, besides ATM, Nbs1, the gene product involved in the Nijmegen Breakage Syndrome (NBS), a disorder that shares with AT chromosome fragility, radiosensitivity and radioresistant DNA synthesis. While in normal cells Chk2 undergoes a time-dependent increased phosphorylation and activation, in NBS cells these events are impaired. These defects can be complemented by the reintroduction of wild type copy of Nbs1 gene, but not by a deletion mutant encoding for a protein unable to form a complex with Mre11 and Rad50 in the nucleus. These results underline the main role of Nbs1 complex in ATM-Chk2 pathway in response to DNA damage and suggest that some defects in NBS and AT cells may result from defective Chk2 activation.

Since Nbs1 and ATM are involved in DNA breaks resolution, I investigated the lesion specificity of Chk2 response. Normal lymphoblastoid cells were exposed to different DNA damaging agents and proteins modifications and activation were evaluated. I demonstrated that ATM and Chk2 respond respectively to a low (<3) and a higher (>19) amount of double strand breaks per cell. Furthermore Chk2 activation was detectable only in response to double strand breaks occurrence but not to single strand breaks or lesions induced by UV or hydroxyurea. These data underline the specificity and the different sensitivity of DNA damage response components.

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