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Role of DNA-PKcs in DNA damage response and cell cycle regulation
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
(English)Manuscript (preprint) (Other academic)
National Category
Cell and Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-265136OAI: oai:DiVA.org:uu-265136DiVA: diva2:862581
Note

The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is an essential component of non-homologous end joining (NHEJ) which is the major DSB repair pathway in mammalian cells. We have previously reported that suppression of DNA-PKcs sensitize cells to radiation without affecting repair. In the present study we used synchronized cells to unmask potential roles of DNA-PKcs in specific cell-cycle phases. siRNA was used to deplete DNA-PKcs to 10-15 % of normal levels and cell cycle progression and DSB repair was observed in synchronized cells irradiated at different cell cycle phases. Surprisingly, cells irradiated in G2 phase showed similar p-H3 frequency after 72 h as unirradiated control cells, irrespective of their DNA-PKcs status. When cells were irradiated in G1/S phase, a significant increase of mitotic cells in siDNA-PKcs treated cells was seen 72 h later. Further, irradiation in G1/ S phase caused initially (0-12h) the same number of DSB (53BP1 foci), however, over time (>24h) 53BP1 foci remained at relatively high levels in DNA-PKcs depleted cells, indicating presence of unrepaired DSB in the following G1. This suggest that DNA-PKcs has an important regulatory role in G1/S and a key function in mitosis.

Available from: 2015-10-22 Created: 2015-10-22 Last updated: 2016-01-13
In thesis
1. Radiation response in human cells: DNA damage formation, repair and signaling
Open this publication in new window or tab >>Radiation response in human cells: DNA damage formation, repair and signaling
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Ionizing radiation induces a range of different DNA lesions. In terms of mutation frequency and mammalian cell survival, the most critical of these lesions is the DNA double-strand break (DSB). DSB left unrepaired or mis-repaired may result in chromosomal aberrations that can lead to permanent genetic changes or cell death. The complexity of the DNA damage and the capacity to repair the DSB will determine the fate of the cell. This thesis focuses on the DNA damage formation, repair and signaling after irradiation of human cells.

Radiation with high linear energy transfer (LET) produces clustered damaged sites in the DNA that are difficult for the cell to repair. Within these clustered sites, non-DSB lesions are formed that can be converted into a DSB and add to the damage complexity and affect DSB repair and the measurement. Heat-labile sites in DNA are converted into DSB at elevated temperatures. We show that heat-released DSB are formed post-irradiation with high-LET ions and increase the initial yield of DSB by 30%-40%, which is similar to yields induced by low-LET radiation.

DNA-PKcs, a central player in non-homologous end-joining (NHEJ), the major mammalian DSB repair pathway, has been found to be both up- and downregulated in different tumor types. In Paper II we show that low levels of DNA-PKcs lead to extreme radiosensitivity but, surprisingly, had no effect on the DSB repair. However, the fraction of cells in G2/M phase increased two-fold in cells with low levels of DNA-PKcs. The study continued in Paper IV, where cells were synchronized to unmask potential roles of DNA-PKcs in specific cell cycle phases. Irradiation of DNA-PKcs suppressed cells in the G1/S phase caused a delay in cell cycle progression and an increase in accumulation of G2 cells. Further, these cells showed defects in DNA repair, where a significant amount of 53BP1 foci remained after 72 h. This further strengthens the hypothesis that DNA-PKcs has a role in regulation of mitotic progression.

Several cellular signaling pathways are initiated in response to radiation. One of these downstream signaling proteins is AKT. We identified an interaction between DNA-PKcs and AKT. Knockouts of both AKT1 and AKT2 impaired DSB rejoining after radiation and low levels of DNA-PKcs increased radiosensitivity and decreased DNA repair further.  

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2015. 52 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1157
Keyword
DNA damage, DNA repair, DSB, NHEJ, DNA-PK, ionizing radiation, heat-labile sites
National Category
Other Basic Medicine Cell and Molecular Biology Cell Biology
Research subject
Biomedical Radiation Science
Identifiers
urn:nbn:se:uu:diva-265137 (URN)978-91-554-9397-4 (ISBN)
Public defence
2015-12-16, Rudbecksalen, Rudbecklaboratoriet, Dag Hammarskjölds v 20, Uppsala, 14:00 (English)
Opponent
Supervisors
Available from: 2015-11-24 Created: 2015-10-22 Last updated: 2016-01-13

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