Year: 2011
Type: article
Abstract: Ataxia telangiectasia-mutated (ATM) is a serine/threonine protein kinase that has critical functions in the cellular responses to DNA damage, including cell cycle checkpoint activation and DNA repair. Since ataxia telangiectasia individuals, who have homozygous mutations in ATM, are exquisitely radiosensitive there is considerable interest in inhibiting the kinase activity of ATM to increase the efficacy of targeted radiotherapy. In this dissertation work, I sought to understand the cellular responses to radiation when ATM kinase activity is transiently inhibited using the small molecule ATM kinase inhibitor KU55933. During my PhD, our laboratory has shown that transient ATM kinase inhibition one hour post-irradiation results in radiosensitization, increased chromosome aberrations and abrogation of sister chromatid exchange. I contributed to these findings by showing that the cellular radiosensitization seen in H460 cells with kinase-inhibited ATM was identical to that seen when ATM protein was disrupted using siRNA prior to the insult. In addition, I demonstrated that 15 minutes of ATM kinase activity post-irradiation is sufficient to trigger the G2/M cell cycle checkpoint, and that subsequent transient inhibition of ATM with KU55933 does not affect recovery from this checkpoint. To gain a more global view of the functional consequences of kinase-inhibited ATM following irradiation, I utilized a SILAC-based tandem mass spectrometry approach, combined with a subcellular fractionation protocol, to determine ATM kinase-dependent spatial proteome dynamics in response to radiation-induced DNA damage. Analysis of the chromatin-associated proteome revealed that the retention of 53BP1 at chromatin is decreased when the kinase activity of ATM is inhibited following ionizing radiation (IR). Using fluorescence recovery after photobleaching in live cells, I determined that the stability of IR-induced GFP-53BP1 foci is decreased when the kinase activity of ATM is inhibited following IR. These results provide a roadmap for understanding ATM kinase-dependent spatial protein dynamics in response to DNA damage.
Author: Serah Choi
Language: English
Topic: DNA Repair Mechanisms
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W1032394645