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(PP358) Live cell imaging of DNA-PK activation and localization at DSB sites induced by microbeam-laser irradiation.
Uematsu, Naoya*,1, Yajima, Hirohiko*,1, Chen, Benjamin1, Chen, David1, 1 Division of Molecular Radiation Biology, Department of Dadiation Oncology, Dallas, TX, US
ABSTRACT- DNA-dependent protein kinase (DNA-PK), consisting of Ku and DNA-PKcs subunits, is the key component of the non-homologous end-joining (NHEJ) pathway of DNA double strand breaks (DSBs) repair. We have previously reported that ionizing radiation induces DNA-PKcs phosphorylation at threonine 2609 (T2609) as well as serine 2056 (S2056) and demonstrated that phosphorylation at these two sites is required for NHEJ-mediated DSB repair. To investigate the in vivo dynamics of DNA-PKcs upon DNA damage, we carried out live cell imaging analysis using DNA-PKcs mutant V3 cell complemented with yellow fluorescent protein (YFP)-tagged DNA-PKcs. Localized DSBs were induced by 365-nm microbeam-laser irradiation and localization of YFP-DNA-PKcs to regions of induced DSBs was observed within seconds after irradiation. In addition, phosphorylation of T2609 and S2056 sites (by immunofluorescence approach on fixed cells) was also observed at DSBs immediately. These results suggesting that, in addition to its normal DSB repair activity, DNA-PKcs could also recognize DNA damage and may play an important role in sensing and signaling of the DSBs. In addition, we will further analyze DNA damage induced DNA-PK activation as a function of cell cycle in live cells. (This work is supported by NIH and NASA)
Key words: DNA-PK, doble strand break repair, microbeam laser, live cell imaging
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