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Measurement of DNA-...
Measurement of DNA-Dependent Protein Kinase Phosphorylation Using Flow Cytometry Provides a Reliable Estimate of DNA Repair Capacity
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- Abramenkovs, Andris (författare)
- Uppsala universitet,Medicinsk strålningsvetenskap
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- Stenerlöw, Bo (författare)
- Uppsala universitet,Medicinsk strålningsvetenskap
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(creator_code:org_t)
- RADIATION RESEARCH SOC, 2017
- 2017
- Engelska.
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Ingår i: Radiation Research. - : RADIATION RESEARCH SOC. - 0033-7587 .- 1938-5404. ; 188:6, s. 597-604
- Relaterad länk:
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https://urn.kb.se/re...
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https://doi.org/10.1...
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Abstract
Ämnesord
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- 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.
Ämnesord
- NATURVETENSKAP -- Biologi -- Biofysik (hsv//swe)
- NATURAL SCIENCES -- Biological Sciences -- Biophysics (hsv//eng)
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