Key Event Title
|Level of Biological Organization|
Key Event Components
Key Event Overview
AOPs Including This Key Event
|AOP Name||Role of event in AOP|
|topoisomerase II poisons, infant leukaemia||KeyEvent|
Key Event Description
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 ans 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
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 atserine 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
DSB occurs in eukaryotic and procaryoytic cells. There is good evidence for conservativism of DSB processing pathways in human cells (Gravel et al. 2008).
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Stenerlöw B., Karin H. Karlsson, Brian Cooper, Björn 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