Expression of the Ku70 subunit (XRCC6) and protection from low dose ionizing radiation during zebrafish embryogenesis

Abstract

The Ku70 protein, a product of the XRCC6 gene, is a component of the nonhomologous end-joining (NHEJ) pathway of DNA repair, which protects cells from the effects of radiation-induced DNA damage. Although the spatial expression of Ku70 during vertebrate embryogenesis has not been described, DNA repair proteins are generally considered to be "housekeeping" genes, which are required for radioprotection in all cells. Here, we report the cloning and characterization of the zebrafish Ku70 ortholog. In situ hybridization and RT-PCR analyses demonstrate that Ku70 mRNA is maternally provided and expressed uniformly among embryonic blastomeres. Later during embryogenesis, zygotically transcribed Ku70 mRNA specifically accumulates in neural tissue, including the retina and proliferative regions of the developing brain. In the absence of genotoxic stress, morpholino-mediated knockdown of Ku70 expression does not affect zebrafish embryogenesis. However, exposure of Ku70 morpholino-injected embryos to low doses of ionizing radiation leads to marked cell death throughout the developing brain, spinal cord, and tail. These results suggest that Ku70 protein plays a crucial role in protecting the developing nervous system from radiation-induced DNA damage during embryogenesis. KeywordsKu protein; Ku70; XRCC6; embryo; ionizing radiation; nonhomologous end-joining; DNA repair; TUNEL; apoptosis; zebrafish The physical interaction of ionizing radiation with duplex DNA results in double-strand breaks (DSBs), which are one of the most potent types of DNA lesion. In proliferating cells, the cytotoxic effects of DSBs can be acute or latently manifest as chromosomal translocations and other genomic aberrations [1]. Natural sources of DSBs include ionizing radiation and, to a lesser extent, byproducts of normal metabolic processes, such as reactive oxygen species. To mitigate the biological effects of DSBs, organisms have developed protective strategies that sense and rapidly repair this type of DNA damage. Following DSB induction, chromosomal integrity is typically reestablished either by the nonhomologous end-joining (NHEJ) or homologous recombination pathways of DNA repair (reviewed in [2,3]).The NHEJ pathway of DSB repair requires the function of at least five genes: XRCC5, which encodes the Ku80 protein [4][5][6], XRCC6, which encodes the Ku70 protein [7][8][9], LIG4 *Corresponding author: IMMAG, CB-2803, Medical College of Georgia,1120 15th Street, Augusta, GA 30912. Phone: (706) 721-8760. Fax: (706) 721-8752. E-mail: dkozlowski@mcg.edu Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. ...

Genotoxicity of acute and chronic gamma‐irradiation on zebrafish cells and consequences for embryo development

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