Two major DNA repair pathways, nonhomologous end-joining (NHEJ) and homologous recombination (HR), repair doublestranded DNA breaks (DSBs) in all eukaryotes. Additionally, several alternative end-joining pathways (or subpathways) have been reported that characteristically use short-sequence homologies at the DNA ends to facilitate joining. How a cell chooses which DNA repair pathway to use (at any particular DSB) is a central and largely unanswered question. For one type of DSB, there is apparently no choice. DSBs mediated by the lymphocyte-specific recombination activating gene (RAG) endonuclease are repaired virtually exclusively by NHEJ. Here we demonstrate that non-RAG-mediated DSBs can be similarly forced into the NHEJ pathway by physical association with the RAG endonuclease.VDJ recombination ͉ DNA-dependent protein kinase V DJ recombination is the molecular mechanism that provides for limitless antigen receptor diversity in developing lymphocytes by assembling immune receptor genes from their disparate component gene segments during lymphocyte development (1). Recombination is initiated by lymphocyte-specific recombination activating genes (RAG1/RAG2) (2, 3) generating double-stranded DNA breaks (DSBs) adjacent to immune receptor coding segments (3). Because introducing DSBs into one's genome can have dire consequences, VDJ recombination is highly regulated. Regulation is achieved by several mechanisms: (i) cell stage-specific expression of RAG mRNA, (ii) targeted degradation of RAG2 at the G 1 /S border mediated by RAG2's C terminus, and (iii) limited access of recombination signal sequences (RSSs) in RAG-expressing cells (4-9). Although two major double-stranded break repair pathways exist in higher eukaryotes, homologous recombination (HR) (errorfree) and nonhomologous end-joining (NHEJ) pathways (errorprone), RAG-mediated breaks are resolved exclusively by NHEJ (10). The error-prone nature of NHEJ is beneficial during VDJ recombination because its inherent imprecision contributes substantially to the diversification of antigen receptor exons generated by VDJ recombination.It is unclear how RAG breaks are restricted to the NHEJ pathway. Several mechanisms may contribute to this restriction. Limiting RAG to G 0 /G 1 may facilitate repair by NHEJ because it is the most active pathway during G 0 /G 1 (4-6). This mechanism alone does not explain the exclusive repair of VDJ DSBs by NHEJ because RAG mutants that lack cell cycle regulation are still absolutely dependent on intact NHEJ for repair. Previous work has suggested that the RAG complex might shepherd recombination intermediates into the NHEJ pathway (11), although a specific mechanism to provide for this RAG function has not been elucidated.A useful method to study VDJ recombination is to assess recombination of plasmid substrates introduced into cultured cells (the Gellert assay), a method described almost two decades ago (12). This assay (still widely used) is the approach used by Taccioli et al. (10) to make their groundbreaking observation that V...