Alternative Pathways for the Repair of RAG-Induced DNA Breaks

Department of Medicine, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021, USA.
Molecular and Cellular Biology (Impact Factor: 4.78). 01/2006; 26(1):131-9. DOI: 10.1128/MCB.26.1.131-139.2006
Source: PubMed


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 >or=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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    • "Thousands of SSBs naturally occur per day in human cells, generally without deleterious consequences (17). The concept of harnessing the benign nature of nicks for stimulation of homologous recombination has been previously suggested in the context of theoretical models and recombination induced by RAG proteins (18–21). In addition, homing endonucleases have been demonstrated to stimulate HDR when converted to nickases (22–27). "
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    • "DNA-PKcs, the catalytic subunit of DNA-PK, orchestrates NHEJ in response to DSBs. It is also critical in V(D)J recombination, and is essential for effective mammalian telomeric end-capping function (Bailey et al., 1999; Lieber, 1999; Meek et al., 2004; Dudley et al., 2005; Weinstock and Jasin, 2006; Zhang et al., "
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    • "In this scenario NHEJ can repair breaks by joining non-contiguous chromosomal regions, resulting in a chromosomal translocation (Weinstock et al. 2006). As opposed to NHEJ, accurate HR does not contribute to translocation events stemming from the presence of multiple DSBs induced by endonucleases (Weinstock et al. 2006). A perhaps even more complicated array of reactions is necessary for HR repair of a DSB. "
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    ABSTRACT: DNA double-strand breaks (DSBs) occur in response to both endogenous and exogenous genotoxic stress. Inappropriate repair of DSBs can lead to either loss of viability or to chromosomal alterations that increase the likelihood of cancer development. In strong support of this assertion, many cancer predisposition syndromes stem from germline mutations in genes involved in DNA DSB repair. Among the most prominent of such tumor suppressor genes are the Breast Cancer 1 and Breast Cancer 2 genes (BRCA1 and BRCA2), which are mutated in familial forms of breast and ovarian cancer. Recent findings implicate BRCA1 as a central component of several distinct macromolecular protein complexes, each dedicated to distinct elements of DNA DSB repair and tumor suppression. Emerging evidence has shed light on some of the molecular recognition processes that are responsible for targeting BRCA1 and its associated partners to DNA and chromatin directly flanking DSBs. These events are required for BRCA1-dependent DNA repair and tumor suppression. Thus, a detailed temporal and spatial knowledge of how breaks are recognized and repaired has profound implications for understanding processes related to the genesis of malignancy and to its treatment.
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