Aberrantly resolved RAG-mediated DNA breaks in Atm-deficient lymphocytes target chromosomal breakpoints incis

Abstract

Canonical chromosomal translocations juxtaposing antigen receptor genes and oncogenes are a hallmark of many lymphoid malignancies. These translocations frequently form through the joining of DNA ends from double-strand breaks (DSBs) generated by the recombinase activating gene (RAG)-1 and -2 proteins at lymphocyte antigen receptor loci and breakpoint targets near oncogenes. Our understanding of chromosomal breakpoint target selection comes primarily from the analyses of these lesions, which are selected based on their transforming properties. RAG DSBs are rarely resolved aberrantly in wild-type developing lymphocytes. However, in ataxia telangiectasia mutated (ATM)-deficient lymphocytes, RAG breaks are frequently joined aberrantly, forming chromosomal lesions such as translocations that predispose (ATM)-deficient mice and humans to the development of lymphoid malignancies. Here, an approach that minimizes selection biases is used to isolate a large cohort of breakpoint targets of aberrantly resolved RAG DSBs in Atm-deficient lymphocytes. Analyses of this cohort revealed that frequently, the breakpoint targets for aberrantly resolved RAG breaks are other DSBs. Moreover, these nonselected lesions exhibit a bias for using breakpoints in cis, forming small chromosomal deletions, rather than breakpoints in trans, forming chromosomal translocations.ataxia telangiectasia mutated ͉ chromosomal translocation ͉ DNA double-strand break repair ͉ V(D)J recombination D ouble-strand breaks (DSBs) in DNA are generated by genotoxic agents and cellular endonucleases as intermediates in several important physiologic processes including V(D)J recombination, Ig class switch recombination (CSR), DNA replication, gene transcription, and meiosis. DNA DSBs activate a highly conserved cellular response that prevents cell cycle progression, initiates repair of the broken DNA ends, and promotes apoptosis of cells with persistent un-repaired DSBs (1, 2). Broken DNA ends from a single DSB are usually rejoined; however, in some processes, such as V(D)J recombination and CSR, DNA ends arising from two DSBs are joined in a regulated fashion, generating a new gene product (1-4). Broken DNA ends from distinct DSBs can also be joined aberrantly, leading to the formation of potentially dangerous chromosomal lesions such as translocations, deletions, and inversions.All developing lymphocytes generate programmed DNA DSBs during V(D)J recombination, a process that joins variable (V), joining (J), and in some cases, diversity (D) gene segments to generate the second exon of antigen receptor genes (4). The V(D)J recombination reaction is initiated by the recombinase activating gene (RAG)-1 and -2 proteins, which together form the RAG endonuclease (5). RAG introduces DNA DSBs at the border of two recombining gene segments and their flanking RAG recognition sequences, termed recombination signals (RSs) (5). DNA cleavage by RAG occurs after an appropriate RS pair (12/23 compatible) forms a synaptic complex, generating

Molecular Mechanisms Underlying Genomic Instability in Brca-Deficient Cells

“…Translocation partners for double-strand breaks are not strictly limited to closely interacting chromosome domains, but when a large number of doublestrand breaks is present, there is an increased frequency of interchromosomal translocation between partners with higher physical interaction 95 . All deep-sequencing studies to date have shown that double-strand breaks on the same chromosome, particularly those lying nearby on the same chromosome, have the highest rate of joining, and this matches a previous study of the frequency of joining of breaks induced by the V(D)J recombination-activating protein 1 (RAG1) and RAG2 recombinases 96 . Taking these studies together, the primary predictor for whether genes take part in translocations is the frequency with which those genes undergo doublestrand breakage.…”

Ataxia telangiectasia mutated (Atm) and DNA-PKcs kinases have overlapping activities during chromosomal signal joint formation

“…S8). The bias for breakpoint targets at the IgLκ locus, which also undergoes inducible rearrangement in these cells, is reminiscent of what was observed for the aberrant resolution of coding ends in Atm-deficient abl pre-B cells (13,27). (13).…”

End-joining, translocations and cancer

“…One possibility is that these recurrent translocations arise at no more common frequency than any other translocation, but are selected on the basis of their potential to drive survival and proliferation of the cancer cell. An additional, long-standing hypothesis is that recurrent translocations arise because the translocation partners are in particularly close proximity in the nuclei of cells from the affected tissue 91, 92, 93 . Chromosome Conformation Capture has been used to measure genomic interactions, and combining this technique with deep sequencing has recently enabled the measurement at base-pair resolution of how closely genomic loci interact.…”