Abstract:Non-homologous end-joining (NHEJ) of DNA double-strand breaks (DSBs) is mediated by two protein complexes comprising Ku80/Ku70/DNA-PKcs/Artemis and XRCC4/LigaseIV/XLF. Loss of Ku or XRCC4/LigaseIV function compromises the rejoining of radiation-induced DSBs and leads to defective V(D)J recombination. In this study, we sought to define how XRCC4 and Ku80 affect NHEJ of site-directed chromosomal DSBs in murine fibroblasts. We employed a recently developed reporter system based on the rejoining of I-SceI endonucl… Show more
“…4A). Schulte-Uentrop et al (35) reported similar results with Ku80-deficient mouse cells; efficient end-joining activity was observed in both WT and Ku80-deficient mouse cells after introducing DSBs enzymatically. However, deletions at the break site in Ku80-deficient mouse cells were significantly longer than in WT mouse cells.…”
“…4A). Schulte-Uentrop et al (35) reported similar results with Ku80-deficient mouse cells; efficient end-joining activity was observed in both WT and Ku80-deficient mouse cells after introducing DSBs enzymatically. However, deletions at the break site in Ku80-deficient mouse cells were significantly longer than in WT mouse cells.…”
“…The NHEJ reporter plasmid pPHW1 was described previously (9,32). pPHW1 was linearized with PvuI and transfected into HEK293 cells using FuGENE 6 (Roche Applied Science).…”
Section: Cell Line and Plasmid Constructionmentioning
confidence: 99%
“…The structure of the NHEJ substrate and the strategy to measure NHEJ were described previously (9,32,33) and are depicted in Rejoining of the double-stranded ends by either precise C-NHEJ or error-prone D-NHEJ will reconstitute translation of the gpt ORF, permitting cell survival under XHATM selection and detection of recombinants as colonies grown in XHATM selection medium. Alternatively, error-prone D-NHEJ at either of the single I-SceI sites can also disrupt ATG ART , allowing again for selection of recombinants with XHATM.…”
Section: Strategy and Generation Of The Hek293/pphw1 Cell Line-mentioning
confidence: 99%
“…In contrast, NHEJ is active throughout the cell cycle and requires little or no DNA homology during repair; thus, it is traditionally considered an error-prone repair pathway (6,7). However, accumulating evidence from recent studies suggests that there exists an error-free NHEJ subpathway (8,9).…”
“…Moreover, transfection of Ku deficient or proficient cells with variants of specifically designed substrate DNAs (with natural AP site or its AP lyase-resistant analog or without AP site) followed by PCR amplification of joining products and subsequent restriction analysis of amplicons fully confirmed the necessity of Ku antigen AP lyase activity for removal of near-end AP sites. Altogether the results obtained in vitro and in vivo testify to use of the 5′-dRP/AP lyase activity of Ku antigen for the excision of near-end abasic sites and explain higher radiosensitivity of mammalian cells deficient in Ku antigen, which is indispensable for classical NHEJ (Schulte-Uentrop et al, 2008). It is worthy of notice, that the same mechanism of AP site cleavage is used by two unrelated DNA repair systems and the suitable positioning of AP sites relative to active site nucleophiles is indispensable for efficient catalysis.…”
Section: Identification Of Ku80 Subunit Of Human Ku Antigen As a Protmentioning
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