title: Alternative pathways for the repair of RAG-induced DNA breaks.
abstract: 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.
authors.affiliation: Department of Medicine, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021, USA.
authors.forename: David M
authors.initials: DM
authors.lastname: Weinstock
authors.forename: Maria
authors.initials: M
authors.lastname: Jasin
mesh_terms: D000818:Animals; D019457:Chromosome Breakage; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004260:DNA Repair / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004622:Embryo, Mammalian / Q000166:cytology / Q000378:metabolism; D005803:Genes, Immunoglobulin / Q000235:genetics; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D011995:Recombination, Genetic / Q000235:genetics; D013234:Stem Cells / Q000378:metabolism*; D045567:VDJ Recombinases / Q000235:genetics / Q000378:metabolism*
chemical_list: D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; C499308:Rag2 protein, mouse; C064658:RAG-1 protein; D004247:DNA; D045567:VDJ Recombinases