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Measurement of DNA-Dependent Protein Kinase Phosphorylation Using Flow Cytometry Provides a Reliable Estimate of DNA Repair Capacity
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
2017 (English)In: Radiation Research, ISSN 0033-7587, E-ISSN 1938-5404, Vol. 188, no 6, p. 597-604Article in journal (Refereed) Published
Abstract [en]

Uncontrolled generation of DNA double-strand breaks (DSBs) in cells is regarded as a highly toxic event that threatens cell survival. Radiation-induced DNA DSBs are commonly measured by pulsed-field gel electrophoresis, microscopic evaluation of accumulating DNA damage response proteins (e.g., 53BP1 or gamma-H2AX) or flow cytometric analysis of gamma-H2AX. The advantage of flow cytometric analysis is that DSB formation and repair can be studied in relationship to cell cycle phase or expression of other proteins. However, gamma-H2AX is not able to monitor repair kinetics within the first 60 min postirradiation, a period when most DSBs undergo repair. A key protein in non-homologous end joining repair is the catalytic subunit of DNA-dependent protein kinase. Among several phosphorylation sites of DNA-dependent protein kinase, the threonine at position 2609 (T2609), which is phosphorylated by ataxia telangiectasia mutated (ATM) or DNA-dependent protein kinase catalytic subunit itself, activates the end processing of DSB. Using flow cytometry, we show here that phosphorylation at T2609 is faster in response to DSBs than gamma-H2AX. Furthermore, flow cytometric analysis of T2609 resulted in a better representation of fast repair kinetics than analysis of gamma-H2AX. In cells with reduced ligase IV activity, and wild-type cells where DNA-dependent protein kinase activity was inhibited, the reduced DSB repair capacity was observed by T2609 evaluation using flow cytometry. In conclusion, flow cytometric evaluation of DNA-dependent protein kinase T2609 can be used as a marker for early DSB repair and gives a better representation of early repair events than analysis of gamma-H2AX.

Place, publisher, year, edition, pages
RADIATION RESEARCH SOC , 2017. Vol. 188, no 6, p. 597-604
National Category
Biophysics
Identifiers
URN: urn:nbn:se:uu:diva-343567DOI: 10.1667/RR14693.1ISI: 000416744600001OAI: oai:DiVA.org:uu-343567DiVA, id: diva2:1187012
Funder
Swedish Cancer SocietySwedish Radiation Safety AuthorityAvailable from: 2018-03-02 Created: 2018-03-02 Last updated: 2018-03-02Bibliographically approved

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Abramenkovs, AndrisStenerlöw, Bo

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