RESUMO
Non-homologous end joining (NHEJ), the major pathway of double-strand break (DSB) repair in mammalian cells, comprises two subpathways: one that requires the three core factors Ku70/80, DNA-PKcs and XRCC4/LigIV (DNA-PK-dependent NHEJ) and the other that is independent of these factors. Using a cell-free NHEJ assay, we have investigated the ability of three Chinese hamster ovary (CHO) mutants deficient in Ku80 (xrs6), DNA-PKcs (XR-C1) and XRCC4 (XR-1) in comparison with CHO-K1 wild-type cells to rejoin non-compatible DSB ends. Both NHEJ efficiency and fidelity are strongly reduced in the mutants with xrs6 and XR-1 exhibiting the strongest reduction and XR-C1 displaying a phenotype intermediate between the wild-type and the other two mutants indicating a non-essential but facilitating role of DNA-PKcs in NHEJ. The decrease in fidelity in the mutants is expressed by an increase of deletion junctions formed at microhomologies (microhom) near the DSB (microhomology-mediated non-homologous end joining: microhomNHEJ). Using a novel microhomNHEJ assay, we show that microhom regions of 6-10 bp that are located directly at the DSB termini strongly enhance the mutagenic microhomNHEJ reaction even in the wild type. Due to its error proneness, DNA-PK-independent microhomNHEJ may actively promote genome instability. It will, therefore, be of increasing importance to examine NHEJ fidelity in the context with tumorigenesis and cellular senescence for which we here provide two efficient and reliable tools.
Assuntos
Quebras de DNA de Cadeia Dupla , Enzimas Reparadoras do DNA/deficiência , Reparo do DNA/fisiologia , Proteína Quinase Ativada por DNA/deficiência , Proteínas de Ligação a DNA/deficiência , Animais , Antígenos Nucleares/genética , Western Blotting , Células CHO , Extratos Celulares , Clonagem Molecular , Cricetinae , Cricetulus , Reparo do DNA/genética , Enzimas Reparadoras do DNA/genética , Proteína Quinase Ativada por DNA/genética , Proteínas de Ligação a DNA/genética , Eletroforese em Gel de Ágar , Imuno-Histoquímica , Cinética , Autoantígeno Ku , Testes de Mutagenicidade/métodos , Oligonucleotídeos/genéticaRESUMO
Double-strand breaks (DSBs) in genomic DNA are induced by ionizing radiation or radiomimetic drugs, but they also occur spontaneously during the cell cycle at quite significant frequencies. In vertebrate cells, nonhomologous DNA end joining (NHEJ) is considered the major pathway of DSB repair. NHEJ is able to rejoin two broken DNA termini directly end-to-end irrespective of sequence and structure. Genetic studies in various radiosensitive and DSB repair-deficient hamster cell lines have yielded insights into the factors involved in NHEJ. Studies in cell-free systems derived from Xenopus eggs and mammalian cells have allowed the dissection of the underlying mechanisms. In the present chapter, we describe a protocol for the preparation of whole cell extracts from mammalian cells and a plasmid-based in vitro assay that permits the easy analysis of the efficiency and fidelity of DSB repair via NHEJ in different cell types.
Assuntos
Sistema Livre de Células , Reparo do DNA , Recombinação Genética , Animais , Sequência de Bases , Células CHO , Extratos Celulares/química , Cricetinae , Cricetulus , DNA/análise , DNA/química , Dano ao DNA , Humanos , Dados de Sequência Molecular , Plasmídeos/genéticaRESUMO
In mammalian cells, nonhomologous DNA end joining (NHEJ) is considered the major pathway of double-strand break (DSB) repair. Rejoining of DSB produced by decay of (125)I positioned against a specific target site in plasmid DNA via a triplex-forming oligonucleotide (TFO) was investigated in cell-free extracts from Chinese hamster ovary cells. The efficiency and quality of NHEJ of the "complex" DSB induced by the (125)I-TFO was compared with that of "simple" DSB induced by restriction enzymes. We demonstrate that the extracts are indeed able to rejoin (125)I-TFO-induced DSB, although at approximately 10-fold decreased efficiency compared with restriction enzyme-induced DSB. The resulting spectrum of junctions is highly heterogeneous exhibiting deletions (1-30 bp), base pair substitutions, and insertions and reflects the heterogeneity of DSB induced by the (125)I-TFO within its target site. We show that NHEJ of (125)I-TFO-induced DSB is not a random process that solely depends on the position of the DSB but is driven by the availability of microhomology patches in the target sequence. The similarity of the junctions obtained with the ones found in vivo after (125)I-TFO-mediated radiodamage indicates that our in vitro system may be a useful tool to elucidate the mechanisms of ionizing radiation-induced mutagenesis and repair.