| Abstract: |
Two major complementary double-strand break (DSB) repair pathways exist in vertebrates, homologous recombination (HR), which involves Rad54, and non-homologous end-joining, which requires the DNA-dependent protein kinase (DNA-PK). DNA-PK comprises a catalytic subunit (DNA-PKcs) and a DNA-binding Ku70 and Ku80 heterodimer. To define the activities of individual DNA-PK components in DSB repair, we targeted the DNA-PKcs gene in chicken DT40 cells. DNA-PKcs deficiency caused a DSB repair defect that was, unexpectedly, suppressed by KU70 disruption. We have shown previously that genetic ablation of Ku70 confers RAD54-dependent radioresistance on S-G2 phase cells, when sister chromatids are available for HR repair. To test whether direct interference by Ku70 with HR might explain the Ku70-/-/DNA-PKcs -/-/- radioresistance, we monitored HR activities directly in Ku- and DNA-PKcs-deficient cells. The frequency of intrachromosomal HR induced by the I-SceI restriction enzyme was increased in the absence of Ku but not of DNA-PKcs. Significantly, abrogation of HR activity by targeting RAD54 in Ku 70-/- or DNA-PKcs-/-/- cells caused extreme radiosensitivity, suggesting that the relative radioresistance seen with loss of Ku70 was because of HR-dependent repair pathways. Our findings suggest that Ku can interfere with HR-mediated DSB repair, perhaps competing with HR for DSB recognition. |
| Keywords: |
unclassified drug; dna binding protein; exon; genetics; dna-binding proteins; exons; nonhuman; genetic analysis; proteins; animal cell; mouse; animal; metabolism; mice; dna damage; gene; homologous recombination; gene targeting; cell cycle; cell cycle s phase; dna repair; genes; nuclear protein; cell line; gene locus; genotype; protein serine threonine kinase; radiation response; physiology; dose-response relationship, radiation; animalia; chromatid; dna strand breakage; nuclear proteins; vertebrata; blotting, western; dna; double stranded dna; genetic recombination; genetic engineering; gene disruption; protein-serine-threonine kinases; recombination, genetic; western blotting; rad54 protein; radiosensitivity; restriction endonuclease; dimerization; cell cycle g2 phase; dna binding; s phase; protein derivative; g2 phase; nocodazole; chickens; chromatids; cells; dna dependent protein kinase; dna-activated protein kinase; chicken; priority journal; article; gallus gallus; support, non-u.s. gov't; genetic ablation; dna activated protein kinase; dna binding proteins; ku protein, human; protein serine threonine kinases; ku70 gene; ku80 gene
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