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Removal of heat-sensitive clustered damaged DNA sites is independent of double-strand break repair
Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi, Medicinsk strålningsvetenskap.
Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi, Medicinsk strålningsvetenskap.
2018 (Engelska)Ingår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 13, nr 12, artikel-id e0209594Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

DNA double-strand breaks (DSBs) are the most deleterious lesions that can arise in cells after ionizing radiation or radiometric drug treatment. In addition to prompt DSBs, DSBs may also be produced during repair, evolving from a clustered DNA damaged site, which is composed of two or more distinct lesions that are located within two helical turns. A specific type of cluster damage is the heat-sensitive clustered site (HSCS), which transforms into DSBs upon treatment at elevated temperatures. The actual lesions or mechanisms that mediate the HSCS transformation into DSBs are unknown. However, there are two possibilities; either these lesions are transformed into DSBs due to DNA lesion instability, e.g., transfer of HSCS into single-strand breaks (SSBs), or they are formed due to local DNA structure instability, e.g., DNA melting, where two SSBs on opposite strands meet and transform into a DSB. The importance of these processes in living cells is not understood, but they significantly affect estimates of DSB repair capacity. In this study, we show that HSCS removal in human cells is not affected by defects in DSB repair or inhibition of DSB repair. Under conditions where rejoining of prompt DSBs was almost completely inhibited, heat-sensitive DSBs were successfully rejoined, without resulting in increased DSB levels, indicating that HSCS do not transfer into DSB in cells under physiological conditions. Furthermore, analysis by atomic force microscopy suggests that prolonged heating of chromosomal DNA can induce structural changes that facilitate transformation of HSCS into DSB. In conclusion, the HSCS do not generate additional DSBs at physiological temperatures in human cells, and the repair of HSCS is independent of DSB repair.

Ort, förlag, år, upplaga, sidor
2018. Vol. 13, nr 12, artikel-id e0209594
Nationell ämneskategori
Cancer och onkologi
Identifikatorer
URN: urn:nbn:se:uu:diva-374120DOI: 10.1371/journal.pone.0209594ISI: 000454621900032PubMedID: 30592737OAI: oai:DiVA.org:uu-374120DiVA, id: diva2:1281985
Forskningsfinansiär
Cancerfonden, CAN2014/661Cancerfonden, CAN2016/649Strålsäkerhetsmyndigheten, SSM2017-2374Strålsäkerhetsmyndigheten, SSM2018-2181Tillgänglig från: 2019-01-23 Skapad: 2019-01-23 Senast uppdaterad: 2019-03-08Bibliografiskt granskad
Ingår i avhandling
1. Induction and repair of clustered DNA damage sites after exposure to ionizing radiation
Öppna denna publikation i ny flik eller fönster >>Induction and repair of clustered DNA damage sites after exposure to ionizing radiation
2019 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
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.

Ort, förlag, år, upplaga, sidor
Uppsala: Acta Universitatis Upsaliensis, 2019. s. 54
Serie
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1548
Nyckelord
NHEJ, DSB repair, clustered DNA damage, DNA repair, DNA-PKcs, HSCS, Ra-223, ionizing radiation
Nationell ämneskategori
Medicin och hälsovetenskap
Forskningsämne
Medicinsk vetenskap
Identifikatorer
urn:nbn:se:uu:diva-378721 (URN)978-91-513-0591-2 (ISBN)
Disputation
2019-04-29, Rudbecksalen, Rudbecklaboratoriet, Dag Hammarskjölds v 20, Uppsala, 13:00 (Engelska)
Opponent
Handledare
Tillgänglig från: 2019-04-04 Skapad: 2019-03-08 Senast uppdaterad: 2019-05-07

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