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Measurement of Prompt DNA Double-Strand Breaks in Mammalian Cells without Including Heat-Labile Sites: Results for Cells Deficient in Nonhomologous End Joining
Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
2003 In: Radiation Research, ISSN 0033-7587, Vol. 159, 502-510 p.Article in journal (Refereed) Published
Place, publisher, year, edition, pages
2003. Vol. 159, 502-510 p.
Identifiers
URN: urn:nbn:se:uu:diva-95066OAI: oai:DiVA.org:uu-95066DiVA: diva2:169133
Available from: 2006-11-10 Created: 2006-11-10Bibliographically approved
In thesis
1. Role of Non-Homologous End-Joining in Repair of Radiation-Induced DNA Double-Strand Breaks
Open this publication in new window or tab >>Role of Non-Homologous End-Joining in Repair of Radiation-Induced DNA Double-Strand Breaks
2006 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Efficient and correct repair of DNA damage, especially DNA double-strand breaks (DSBs), is vital for the survival of individual cells and organisms. Defects in the DNA repair may lead to cell death or genomic instability and development of cancer.

The repair of DSBs in cell lines with different DSB rejoining capabilities was studied after exposure to ionising radiation. A new cell lysis protocol performed at 0ºC, which prevents the inclusion of non-true DSBs in the quantification of DSBs by pulsed-field gel electrophoresis (PFGE), was developed. Results showed that when the standard protocol at 50ºC was used, 30-40% of the initial yield of DSBs corresponds to artifactual DSBs. The lesions transformed to DSBs during incubation at 50ºC were repaired within 60-90 minutes in vivo and the repair was independent of DNA-PK, XRCC1 and PARP-1.

Non-homologous end-joining (NHEJ) is the major DSB repair pathway in mammalian cells. We show that DSBs are processed into long single-stranded DNA (ssDNA) ends after ≥1 h of repair in NHEJ deficient cells. The ssDNA was formed outside of the G1 phase of the cell cycle and only in the absence of the NHEJ proteins DNA-PK and DNA Ligase IV/XRCC4. The generation of ssDNA had great influence on the quantification of DSBs by PFGE. The standard protocol caused hybridisation of the ssDNA ends, resulting in overestimation of the DSB repair capability in NHEJ deficient cells.

DSBs were also quantified by detection of phosphorylated H2AX (γ-H2AX) foci. A large number of γ-H2AX foci still remaining after 21 h of repair in an NHEJ deficient cell line confirmed the low repair capability determined by PFGE. Furthermore, in normal cells difficulty in repairing clustered breaks was observed as a large fraction of γ-H2AX foci remaining 24 h after irradiation with high-LET ions.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2006. 49 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 194
Keyword
Molecular biology, DNA damage, DNA repair, DSB, NHEJ, DNA-PK, ionising radiation, high-LET, heat-labile sites, PFGE, Molekylärbiologi
Identifiers
urn:nbn:se:uu:diva-7219 (URN)91-554-6702-4 (ISBN)
Public defence
2006-12-02, Rudbecksalen, Rudbecklaboratoriet, Dag Hammarskjölds väg 20, Uppsala, 09:15
Opponent
Supervisors
Available from: 2006-11-10 Created: 2006-11-10Bibliographically approved

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