期刊
MOLECULAR AND CELLULAR BIOLOGY
卷 26, 期 1, 页码 131-139出版社
AMER SOC MICROBIOLOGY
DOI: 10.1128/MCB.26.1.131-139.2006
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资金
- NCI NIH HHS [T32 CA009512, CA09512] Funding Source: Medline
- PHS HHS [54688] Funding Source: Medline
- NATIONAL CANCER INSTITUTE [T32CA009512] Funding Source: NIH RePORTER
RAG1 and RAG2 cleave DNA to generate blunt signal ends and hairpin coding ends at antigen receptor loci in lymphoid cells. During V(D)J recombination, repair of these RAG-generated double-strand breaks (DSBs) by the nonhomologous end-joining (NHEJ) pathway contributes substantially to the antigen receptor diversity necessary for immune system function, although recent evidence also supports the ability of RAG-generated breaks to undergo homology-directed repair (HDR). We have determined that RAG-generated chromosomal breaks can be repaired by pathways other than NHEJ in mouse embryonic stem (ES) cells, although repair by these pathways occurs at a significantly lower frequency than NHEJ. HDR frequency was estimated to be >= 40-fold lower than NHEJ frequency for both coding end and signal end reporters. Repair by single-strand annealing was estimated to occur at a comparable or lower frequency than HDR. As expected, V(D)J recombination was substantially impaired in cells deficient for the NHEJ components Ku70, XRCC4, and DNA-PKcs. Concomitant with decreased NHEJ, RAG-induced HDR was increased in each of the mutants, including cells lacking DNA-PKcs, which has been implicated in hairpin opening. HDR was increased to the largest extent in Ku70(-/-) cells, implicating the Ku70/80 DNA end-binding protein in regulating pathway choice. Thus, RAG-generated DSBs are typically repaired by the NHEJ pathway in ES cells, but in the absence of NHEJ components, a substantial fraction of breaks can be efficiently channeled into alternative pathways in these cells.
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