Spi − selection takes advantage of the restricted growth of the lambda phage in P2 lysogens. A feature of the mutation assay is its ability to efficiently detect certain types of deletions by Spi − (sensitive to P2 interference) selection, as well as point mutations, i.e., base substitutions and frameshifts, by 6-thioguanine selection. In gpt delta mice, about 80 copies of lambda EG10 DNA, which carries red and gam genes, are integrated into each chromosome 17 in a C57BL/6 J background. We previously developed the gpt delta transgenic mouse for the detection of mutations in vivo. Therefore, it appears that DNA-PKcs is only needed to resolve a subset of DSBs and that NHEJ may proceed in a DNA-PKcs-independent, as well as DNA-PKcs-dependent, manner. In contrast, Ku-deficient mice and embryonic stem cells exhibit defects in both coding and signal joint formation. However, scid cells can form signal joints in V(D)J recombination, which involve the joining of the ends created after the excision of intervening DNA during V(D)J recombination and the formation of circular DNA molecules. These mice, and cells derived from them, are hypersensitive to IR, display defects in joining of IR-induced DNA DSBs, and are defective in coding joint formation during V(D)J recombination. ![]() The scid (severe combined immune-deficiency) mice bear a naturally occurring mutation in the DNA-PKcs gene that results in an 83-amino acid truncation of the C-terminal end. However, different proteins are recruited to the site of DNA damage to participate in the repair of DSBs each time, depending on the end configurations i.e., blunt ends, 3′- or 5′-overhands or ends containing modified bases. In general, when DNA DSBs are induced, Ku70/80 proteins bind to the ends and interact with other proteins including DNA PKcs and Artemis for end-resection, DNA polymerase μ and λ for addition of nucleotides, and the DNA ligase IV complex for ligation of the ends. Therefore, NHEJ can be regarded as a double-edged sword while it prevents cell death and gross chromosome rearrangements, it often induces deletions and insertions during ligation of incompatible ends.ĭNA-dependent protein kinase (DNA-PK) consists of three components, the catalytic subunit DNA-PKcs and the heterodimeric Ku70 and Ku80 proteins, and is involved in NHEJ of DNA DSBs and V(D)J recombination. IR and chemical treatments usually induce modified DSBs with ends that are incompatible for direct ligation. ![]() Although defects in NHEJ result in genomic instability and cancer predisposition, NHEJ often leads to deletion mutations, with or without short length insertions, when DNA ends can’t be directly ligated. This is particularly true in non-dividing cells and in G1 cells due to the absence of sister chromatids, the preferred substrate for homologous recombination. However, DSBs induced by ionizing radiation (IR) are mainly repaired through NHEJ pathway. In mammalian cells, DSBs are repaired by the homologous recombination and/or the nonhomologous end-joining (NHEJ) pathways. The repair of DNA double-strand breaks (DSBs) is critical for the maintenance of genomic integrity. Therefore, the contribution of DNA-PKcs to NHEJ may be organ-specific. ConclusionsĭNA-PKcs is unessential for the induction of deletion mutations in the spleen, while it plays a role in this in the brain. Unirradiated scid and WT mice did not exhibit significant differences in MFs in either organ. The MFs in the brain of irradiated scid mice were significantly lower than those in WT mice, i.e., 2.9 ± 1.0 × 10 − 6 versus 5.0 ± 1.1 × 10 − 6 ( P < 0.001), respectively. Irradiation with X-rays significantly enhanced Spi − MF in both organs in the scid and WT mice. The scid and wild-type (WT) gpt delta transgenic mice were irradiated with a single X-ray dose of 10 Gy, and Spi − mutant frequencies (MFs) were determined in the brain and spleen 2 days after irradiation. ![]() To examine the roles of DNA-PKcs in the generation of deletion mutations in vivo, we crossed scid mice with gpt delta transgenic mice for detecting mutations. Severe combined immunodeficient ( scid) mice carry a mutation in the gene encoding DNA-PKcs and are sensitive to ionizing radiation. ![]() DNA-dependent protein kinase (DNA-PK), consisting of a Ku heterodimer (Ku70/80) and a large catalytic subunit (DNA-PKcs), plays an important role in the repair of DNA double-strand breaks via non-homologous end-joining (NHEJ) in mammalian cells.
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