We previously described a fluore Scence-based assay for measuring the frequency of HR at a chromosomal DSB in murine embryonic stem (ES) cells ( Fig. Among the NHEJ pathways, ‘classic’ NHEJ is the most well-studied and involves the heterodimer Ku70/Ku80, the serine/threonine kinase DNA-PKcs, the XRCC4/Ligase IV (Lig4) complex, and Artemis ( Lieber et al., 2003). In NHEJ, the DSB ends may be modified by the addition or deletion of nucleotides prior to ligation, making NHEJ potentially mutagenic as well. The SSA pathway is mutagenic, because the sequence between the repeats is deleted upon the completion of the reaction. By contrast, SSA involves the annealing of complementary single-strand tails formed at repeated sequences and is inhibited by RAD51 ( Stark et al., 2004). the sister chromatid) is used in the reaction ( Johnson and Jasin, 2000) ( Johnson and Jasin, 2000). HR involves the recruitment of the RAD51 protein to the single-strand tails, which promotes a strand invasion reaction (Sung et al., 2004), and eventually leads to the restoration of the initial genomic sequence if an identical template ( e.g. In both HR and SSA, sequences adjacent to a DSB are processed to 3’ single-strand tails. Depending on the context of the DSB, another pathway of repair involving homology can also be used for repair, which is termed single-strand annealing (SSA). The repair of DNA double-strand breaks (DSBs) in mammalian cells occurs by nonhomologous end-joining (NHEJ) or by homologous recombination (HR), a process in which a homologous sequence acts as a repair template. Measuring the nature and frequency of repair of DNA double-strand breaks generated by the I- SceI endonuclease These reporters can be used to assess the effects of genetic background, dominant-negative constructs or physiological conditions on DSB repair in a wide variety of mammalian cellsġ. The individual contributions of NHEJ, HR, and SSA can be quantified using fluore Scence and PCR-based assays after the precise introduction of DSBs either by the I- SceI endonuclease or by the RAG recombinase. This chapter describes the use of reporter substrates for assaying the contributions of these pathways to DSB repair in mammalian cells, in particular murine embryonic stem cells. Mammalian cells have multiple pathways for repairing DSBs, including nonhomologous end-joining (NHEJ), homologous recombination (HR), and single-strand annealing (SSA). DNA damage in the form of double-strand breaks (DSBs) can occur as a result of both exogenous insults, such as ionizing radiation and drug therapies, and normal metabolic processes including V(D)J recombination. Unrepaired or imprecisely repaired DNA can lead to mutagenesis, cell death or malignant transformation. DNA damage repair is essential for the maintenance of genetic integrity in all organisms.
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