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Molecular and Cellular Biology, January 2006, p. 131-139, Vol. 26, No. 1
0270-7306/06/$08.00+0     doi:10.1128/MCB.26.1.131-139.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Alternative Pathways for the Repair of RAG-Induced DNA Breaks

David M. Weinstock1 and Maria Jasin2*

Department of Medicine,1 Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, New York 100212

Received 15 May 2005/ Returned for modification 13 June 2005/ Accepted 13 October 2005

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.

* Corresponding author. Mailing address: Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021. Phone: (212) 639-7438. Fax: (212) 717-3317. E-mail: m-jasin{at}ski.mskcc.org.

Molecular and Cellular Biology, January 2006, p. 131-139, Vol. 26, No. 1
0022-538X/06/$08.00+0     doi:10.1128/MCB.26.1.131-139.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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