title: A double-strand break can trigger immunoglobulin gene conversion.
abstract: All three B cell-specific activities of the immunoglobulin (Ig) gene re-modeling system-gene conversion, somatic hypermutation and class switch recombination-require activation-induced deaminase (AID). AID-induced DNA lesions must be further processed and dissected into different DNA recombination pathways. In order to characterize potential intermediates for Ig gene conversion, we inserted an I-SceI recognition site into the complementarity determining region 1 (CDR1) of the Ig light chain locus of the AID knockout DT40 cell line, and conditionally expressed I-SceI endonuclease. Here, we show that a double-strand break (DSB) in CDR1 is sufficient to trigger Ig gene conversion in the absence of AID. The pattern and pseudogene usage of DSB-induced gene conversion were comparable to those of AID-induced gene conversion; surprisingly, sometimes a single DSB induced multiple gene conversion events. These constitute direct evidence that a DSB in the V region can be an intermediate for gene conversion. The fate of the DNA lesion downstream of a DSB had more flexibility than that of AID, suggesting two alternative models: (i) DSBs during the physiological gene conversion are in the minority compared to single-strand breaks (SSBs), which are frequently generated following DNA deamination, or (ii) the physiological gene conversion is mediated by a tightly regulated DSB that is locally protected from non-homologous end joining (NHEJ) or other non-homologous DNA recombination machineries.
authors.affiliation: IFOM - FIRC Institute of Molecular Oncology Foundation, Via Adamello 16, 20139 Milan, Italy.|Università degli Studi di Milano, Dipartimento di Bioscienze, Via Celoria 26, 20133 Milan, Italy.
authors.forename: Giulia
authors.initials: G
authors.lastname: Bastianello
authors.affiliation: IFOM - FIRC Institute of Molecular Oncology Foundation, Via Adamello 16, 20139 Milan, Italy hiroshi.arakawa@ifom.eu.
authors.forename: Hiroshi
authors.identifier: http://orcid.org/0000-0002-8103-4549
authors.initials: H
authors.lastname: Arakawa
mesh_terms: D000818:Animals; D001402:B-Lymphocytes / Q000166:cytology / Q000276:immunology*; D001483:Base Sequence; D045744:Cell Line, Tumor; D002645:Chickens; D022801:Complementarity Determining Regions / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D003564:Cytidine Deaminase / Q000172:deficiency / Q000235:genetics / Q000276:immunology; D053903:DNA Breaks, Double-Stranded; D053904:DNA Breaks, Single-Stranded; D004260:DNA Repair / Q000276:immunology*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics / Q000378:metabolism; D005785:Gene Conversion; D017578:Immunoglobulin Class Switching; D007147:Immunoglobulin Light Chains / Q000737:chemistry / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D000098830:AICDA (Activation-Induced Cytidine Deaminase)
chemical_list: D022801:Complementarity Determining Regions; D007147:Immunoglobulin Light Chains; D029701:Saccharomyces cerevisiae Proteins; C066166:AI4 protein, S cerevisiae; D015252:Deoxyribonucleases, Type II Site-Specific; D000098830:AICDA (Activation-Induced Cytidine Deaminase); D003564:Cytidine Deaminase