Transient depletion of Ku70 and Xrcc4 by RNAi as a means to manipulate the non-homologous end-joining pathway

• Published in: Journal of biotechnology Vol. 128; no. 2; pp. 246 - 257
• Main Authors: Bertolini, Luciana R., Bertolini, Marcelo, Anderson, Gary B., Maga, Elizabeth A., Madden, Knut R., Murray, James D.
• Format: Journal Article
• Language: English
• Published: Lausanne Elsevier B.V 01-02-2007; Amsterdam Elsevier; New York, NY
• Subjects: Antigens, Nuclear - genetics; Antigens, Nuclear - metabolism; Biological and medical sciences; Biotechnology; DNA Repair - genetics; DNA Repair - radiation effects; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; Fundamental and applied biological sciences. Psychology; Gene Expression Regulation - genetics; Green Fluorescent Proteins; HCT116 Cells; Humans; Ku Autoantigen; Molecular Sequence Data; Non-homologous end joining; Radiation sensitivity; RNA Interference; RNA, Small Interfering - genetics; RNA, Small Interfering - radiation effects; Transfection; Xrcc4; Radiation sensitivity; Ku; RNA interference; Non-homologous end joining; Xrcc4; Sensitivity; Radiation
• ISSN: 0168-1656; 1873-4863
• DOI: 10.1016/j.jbiotec.2006.10.003

Non-homologous end joining (NHEJ) is the major DNA double-strand break (DSB) repair pathway in mammalian cells and is likely responsible for the non-homologous integration of transgenes. In higher eukaryotes, this pathway predominates over the homologous recombination (HR) pathway and therefore may account for the low level of HR events that occur in mammalian cells. We evaluated the effects of transient RNAi-induced down-regulation of key components of the NHEJ pathway in human HCT116 cells. Treatment with siRNA targeting Ku70 and Xrcc4 reduced corresponding protein levels by 80–90% 48
h after transfection, with a return to normal levels by 96
h. Additionally, down-regulation of Ku70 and Xrcc4 resulted in a concomitant depletion of both Ku70 and Ku86 proteins. Biological consequences of transient RNAi-mediated depletion of Ku70 and Xrcc4 included sensitization to γ radiation and a significant decrease in the expression of a linear GFP reporter gene. The results highlight the possibility of a successful means to manipulate the NHEJ pathway by RNAi.