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Extensive ssDNA End Formation at DNA Double-Strand Breaks in Non-Homologous End-Joining Deficient Cells during the S phase
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
2007 (English)In: BMC Molecular Biology, ISSN 1471-2199, Vol. 8, 97- p.Article in journal (Refereed) Published
Abstract [en]

Background: Efficient and correct repair of DNA damage, especially DNA double-strand breaks, is critical for cellular survival. Defects in the DNA repair may lead to cell death or genomic instability and development of cancer. Non-homologous end-joining (NHEJ) is the major repair pathway for DNA double-strand breaks in mammalian cells. The ability of other repair pathways, such as homologous recombination, to compensate for loss of NHEJ and the ways in which contributions of different pathways are regulated are far from fully understood. Results: In this report we demonstrate that long single-stranded DNA (ssDNA) ends are formed at radiation-induced DNA double-strand breaks in NHEJ deficient cells. At repair times >= 1 h, processing of unrejoined DNA double-strand breaks generated extensive ssDNA at the DNA ends in cells lacking the NHEJ protein complexes DNA-dependent protein kinase (DNA-PK) or DNA Ligase IV/XRCC4. The ssDNA formation was cell cycle dependent, since no ssDNA ends were observed in G(1)-synchronized NHEJ deficient cells. Furthermore, in wild type cells irradiated in the presence of DNA-PKcs ( catalytic subunit of DNA-PK) inhibitors, or in DNA-PKcs deficient cells complemented with DNA-PKcs mutated in six autophosphorylation sites (ABCDE), no ssDNA was formed. The ssDNA generation also greatly influences DNA double-strand break quantification by pulsed-field gel electrophoresis, resulting in overestimation of the DNA double-strand break repair capability in NHEJ deficient cells when standard protocols for preparing naked DNA (i.e., lysis at 50 C) are used. Conclusion: We provide evidence that DNA Ligase IV/XRCC4 recruitment by DNA-PK to DNA double-strand breaks prevents the formation of long ssDNA ends at double-strand breaks during the S phase, indicating that NHEJ components may downregulate an alternative repair process where ssDNA ends are required.

Place, publisher, year, edition, pages
2007. Vol. 8, 97- p.
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:uu:diva-95069DOI: 10.1186/1471-2199-8-97ISI: 000252381300001PubMedID: 17963495OAI: oai:DiVA.org:uu-95069DiVA: diva2:169136
Available from: 2006-11-10 Created: 2006-11-10 Last updated: 2010-04-19Bibliographically 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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