API

Event: 1461

Key Event Title

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DNA double-strand break

Short name

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DSB

Biological Context

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Level of Biological Organization
Molecular

Cell term

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Organ term

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Key Event Components

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Process Object Action

Key Event Overview


AOPs Including This Key Event

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AOP Name Role of event in AOP
topoisomerase II binding, infant leukaemia KeyEvent

Stressors

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Taxonomic Applicability

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Life Stages

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Sex Applicability

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Key Event Description

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DNA double-strand breaks (DSB) is formed as a consequence of the production of excision repair breaks opposite each other on the two strands of DNA, and by the production of an excision repair break opposite a DNA daughter-strand gap. DSB are considered to be critical primary lesions in the formation of chromosomal aberrations.

To repare this potentially lethal damage, eukaryotic cells have evolved a variety of repair pathways related to homologous and illegitimate recombination, also called non-homologous DNA end joining (NHEJ), which may induce small scale mutations and chromosomal aberration (Pfeiffer et al. 2000). Repair by NHEJ often leads to small deletions at the site of the DSB and is considered error prone. The second repair mechanism, the Homologous Recombination (HR) is directed by extensive homology in a partner DNA molecule. In mitotic cells NHEJ occurs throughout all phases of the cell cycle, whereas HR is largely restricted to the S and G2 phases when the sister chromatid is available to mediate the repair process (Reynard et al. 2017). Persistent or incorrectly repaired DSBs can result in chromosome loss, deletion, translocation, or fusion, which can lead to carcinogenesis through activation of oncogenes or inactivation of tumor-suppressor genes (Raynard et al.2017). The DSB repair pathways apper to compete for DSBs, but the balance between them differs widely among species, between different cell types of a single species, and during different cell cycle phases of a single cell type. (Shrivastav et al. 2008).

DSBs are induced by agents such as ionizing radiation and chemicals that directly or indirectly damage DNA and are commonly used in cancer therapy (Shrivastav et al. 2008). DSBs also arise during DNA replication when the DNA-polymerase ensemble encounters obstacles such as DNA lesions or unusual DNA structures (Raynard et al 2017). Additional endogenous sources include reactive oxygen species, generated during cellular metabolism, collapsed replication forks and nucleases(Shrivastav et al. 2008) .


How It Is Measured or Detected

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 A very early step in the cellular response to DSBs is the phosphorylation of a histone H2A variant, H2AX, at the sites of DNA damage. H2AX is rapidly phosphorylated (within seconds) at serine 139 when DSBs are introduced into mammalian cells resulting in discrete γ-H2AX (phosphorylated H2AX) foci at the DNA damage sites. H2AX phosphorylation also appearsto be a general cellular response to processes involving DSB intermediates including V(D)J recombination in lymphoid cells and meiotic recombination in mice. Phosphorylation of H2A at serine 139 causes chromatin decondensation and appears to play a critical role in the recruitment of repair or damage-signaling factors to the sites of DNA damage.  DNA DSB staining  based on the phosphorylation of the histone H2A.X at serine 139 in response to DNA damaging agents which cause double strand breaks in cells that are cultured in microtiter plates is a rapid metod for the identification and quantification of the damage (Sealunavov et al.2002).

Microscopic examination of individual mammalian cells embedded in agarose, subjected to electrophoresis, and stained with a DNA-binding dye provides a way of measuring DNA damage and of assessing heterogeinicity in DNA damage within a mixed cell population. (Olive P. et al. 1991).

Pulsed field gel electrophoresis (PFGE) is the main method used for measurement of DNA DSB in mammalian cells (Blocker D et al. 1989 and 1990, Stamato T et al 1990, Ager D et al 1990). Alternatively the DNA is size fractioned in the pulsed-field gel, and the weight fraction of DNA below a certain defined size is measured (Erixo K. et al. 1990, Stenelow B. et al. 1995). An additional method to measure prompt DSBs without including heat-labile sites is also reported (Stenerlow B. et al. 2003).

In vitro assays for topoisomerase II based on the decantation of double strand DNA are extensively reported in Nitiss et al. 2012.

 


Domain of Applicability

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DSB occurs in eukaryotic and procaryoytic cells. There is good evidence for conservativism of DSB processing pathways in human cells (Gravel et al. 2008).


References

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Ager, D. D., W. C. Dewey, K. Gardiner, W. Harvey, R. T. Johnson, and C. A. Waldren. Measurement of radiation-induced DNA double-strand breaks by pulsed-field gel electrophoresis. Radiat. Res 122:181–187.1990

Blöcher, D., M. Einspenner, and J. Zajackowski. CHEF electrophoresis, a sensitive technique for the determination of DNA double-strand breaks. Int. J. Radiat. Biol 56:437–448.1989.

Blöcher, D. In CHEF electrophoresis a linear induction of dsb correspond to a nonlinear fraction of extracted DNA with dose. Int. J. Radiat. Biol 57:7–12.1990

Blöcher, D. In CHEF electrophoresis a linear induction of dsb correspond to a nonlinear fraction of extracted DNA with dose. Int. J. Radiat. Biol 57:7–12.1990.

Erixon, K., B. Cedervall, and R. Lewensohn. Pulsed-field gel electrophoresis for measuring radiation-induced DNA double-strand breaks. Comparison to the method of neutral filter elution. In Ionizing Radiation Damage to DNA: Molecular Aspects (R. Painter and S. Wallace, Eds.), pp. 69–80. UCLA Symposium on Molecular and Cellular Biology, New Series, Vol. 136, Wiley-Liss, New York, 1990.

Gravel S., Chapman JR., magill C., and jackson SP. 2008. DNA helicases Sgs1 and BLM promote DNA double-strand break resectio. Genes & Dev. 22:2767-2772.

Nitiss JL, Soans E, Rogoljina A, Seth A, Mishina M. 2012 Topoisiomerase assays. Current Protoc Pharmacol. Chapter: Unit 3.3.

Olive. PL, Wlodek D., Banath JP. 1991. DNA double-strand break measured in individual cells subjected to gel electrophoresis. Cnancer research. 51, 4671-4676, September 1.

Pfeiffer P., Goedeke W. and Gunter Obe. 2000. mechanisms of DNA double-strand break repair and their potential to induce chromosomal aberrations. Mutagenesis vol15 n 4 289-302.

Raynard S., Niu H. and Sung P. 2017. 2002. DNA double-strand break processing: the beginning of the end. Genes & Dev. 22: 2903-2907.

Shrivastav M, De Haro LP, Nickoloff JA. 2008. Regulation of DNA double-strand break repair pathway choice. Cell Research, 18: 134-147.

Seluanov A, Zhiyong Mao, and Vera Gorbunova. 2002. Analysis of DNA Double-strand Break (DSB) Repair in Mammalian Cells. J Vis Exp. 2010; (43): 2002. Published online 2010 Sep 8. doi:  10.3791/2002 PMCID: PMC3157866

Stamato, T. D. and N. Denko. Asymmetric field inversion gel electrophoresis: A new method for detecting DNA double-strand breaks in mammalian cells. Radiat. Res 121:196–205.1990. 

Stenerlow, B., J. Carlsson, E. Blomquist, and K. Erixon. Clonogenic cell survival and rejoining of DNA double-strand breaks: Comparisons between three cell lines after photon or He ion irradiation. Int. J. Radiat. Biol 65:631–639.1994. 

Stenerlöw B., Karin H. KarlssonBrian CooperBjörn Rydberg 2003 Measurement of Prompt DNA Double-Strand Breaks in Mammalian Cells without Including Heat-Labile Sites: Results for Cells Deficient in Nonhomologous End JoiningRadiation Research 159(4):502-510. https://doi.org/10.1667/0033-7587(2003)159[0502:MOPDDS]2.0.CO;2

 

 

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