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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: 2019-03-08Bibliographically approved
In thesis
1. Induction and repair of clustered DNA damage sites after exposure to ionizing radiation
Open this publication in new window or tab >>Induction and repair of clustered DNA damage sites after exposure to ionizing radiation
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The mechanisms that maintain genomic stability safeguard cells from constant DNA damage produced by endogenous and external stressors. Therefore, this thesis aimed to specifically address questions regarding the requirement and involvement of DNA repair proteins in the repair of various types of radiation-induced DNA damage.

The first aim was to determine whether the phosphorylation of DNA-PKcs, a major kinase involved in non-homologous end joining pathway, can be utilized to score the DNA double-strand break (DSB) content in cells. DNA-PKcs phosphorylated (pDNA-PKcs) at T2609 was more sensitive to the cellular DSB content than ɣH2AX, as analyzed by flow cytometry. Further, pDNA-PKcs at T2609 could discriminate between DSB repair-compromised and normal cells, confirming that the pDNA-PKcs can be used as a DSB repair marker. In paper II, the DSB repair was assessed in cells with reduced levels of DNA-PKcs. The reduction in DNA-PKcs resulted in decreased cell survival and unaffected DSB repair. These results clearly indicate that DNA-PKcs plays an additional role in promoting cell survival in addition to its function in DSB repair.

The second part of the thesis focused on the characterization of complex DNA damage. DNA damage was investigated after exposure to α-particles originating from Ra-223. The Ra-223 treatment induced a nonrandom DSB distribution consistent with damage induced by high-linear energy transfer radiation. The exposure to Ra-223 significantly reduced cell survival in monolayers and 3D cell structures. The last paper unraveled the fate of heat-sensitive clustered DNA damage site (HSCS) repair in cells. HSCS repair was independent of DSB repair, and these lesions did not contribute to the generation of additional DSBs during repair. Prolonged heating of DNA at relatively low temperatures induced structural changes in the DNA that contributed to the production of DNA artifacts.

In conclusion, these results demonstrate that DNA-PKcs can be used to monitor DSB repair in cells after exposure to ionizing radiation. However, the functions of DNA-PKcs are not limited to DSB repair, as it can promote cell survival through other mechanisms. The complexity of the DNA damage produced by high-LET radiation is a major contributor to cell death. However, not all clusters produced in irradiated cells are converted into DSBs during repair.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2019. p. 54
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1548
Keywords
NHEJ, DSB repair, clustered DNA damage, DNA repair, DNA-PKcs, HSCS, Ra-223, ionizing radiation
National Category
Medical and Health Sciences
Research subject
Medical Science
Identifiers
urn:nbn:se:uu:diva-378721 (URN)978-91-513-0591-2 (ISBN)
Public defence
2019-04-29, Rudbecksalen, Rudbecklaboratoriet, Dag Hammarskjölds v 20, Uppsala, 13:00 (English)
Opponent
Supervisors
Available from: 2019-04-04 Created: 2019-03-08 Last updated: 2019-05-07

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Abramenkovs, AndrisStenerlöw, Bo

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