Staggered AID-dependent DNA double strand breaks are the predominant DNA lesions targeted to S mu in Ig class switch recombination.
Class switch recombination (CSR) is the process whereby B cells alter the effector properties of their Ig molecules. Whilst much is known about the cellular regulation of this process, many of the molecular details remain elusive. Recent evidence suggests that CSR involves blunt DNA double strand breaks (dsbs), and that formation of these dsbs requires the function of the activation-induced cytidine deaminase (AID). We sought to characterize the structural properties and kinetics of induction of the DNA lesions associated with CSR. Using ligation-mediated PCR, we found that AID-dependent DNA dsbs were specifically induced in the S mu region of murine B cells stimulated to undergo CSR. While blunt dsbs were detected, they were only a minor species, with staggered breaks being more than an order of magnitude more abundant. In addition, these breaks could be detected at equal frequency at upstream and downstream portions of S mu, and were induced prior to expression of newly switched isotypes. Collectively, these results provide direct evidence that staggered, S mu-targeted AID-dependent dsbs are the predominant DNA lesion associated with CSR, with important implications for the mechanisms by which CSR DNA lesions are made and processed.
Rush
James S
JS
Section of Immunobiology, Howard Hughes Medical Institute and Yale School of Medicine, New Haven, CT, USA.
Fugmann
Sebastian D
SD
Schatz
David G
DG
D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D001402:B-Lymphocytes / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D015139:Blotting, Southern; D002455:Cell Division / Q000276:immunology / Q000502:physiology; D003564:Cytidine Deaminase / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D004247:DNA / Q000302:isolation & purification / Q000378:metabolism; D004249:DNA Damage; D017931:DNA Primers; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D005434:Flow Cytometry; D015870:Gene Expression / Q000276:immunology; D017578:Immunoglobulin Class Switching / Q000235:genetics* / Q000276:immunology / Q000502:physiology; D007072:Immunoglobulin D / Q000276:immunology; D007074:Immunoglobulin G / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics; D007134:Immunoglobulin Switch Region / Q000235:genetics* / Q000276:immunology / Q000502:physiology; D015847:Interleukin-4 / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011995:Recombination, Genetic / Q000235:genetics; D000098830:AICDA (Activation-Induced Cytidine Deaminase)
D000911:Antibodies, Monoclonal; D017931:DNA Primers; D007072:Immunoglobulin D; D007074:Immunoglobulin G; D008070:Lipopolysaccharides; D015847:Interleukin-4; D004247:DNA; D015252:Deoxyribonucleases, Type II Site-Specific; C027440:GATATC-specific type II deoxyribonucleases; D000098830:AICDA (Activation-Induced Cytidine Deaminase); D003564:Cytidine Deaminase