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From the Departments of Medicine and Molecular Biology & Genetics, The Johns Hopkins University School of Medicine, Baltimore Maryland 21205

The process of V(D)J recombination is crucial for regulating the development of B cells and for determining their eventual antigen specificity. Here we assess the developmental regulation of the V(D)J recombinase directly, by monitoring the double-stranded DNA breaks produced in the process of V(D)J recombination. This analysis provides a measure of recombinase activity at immunoglobulin heavy and light chain loci across defined developmental stages spanning the process of B cell development. We find that expression of a complete immunoglobulin heavy chain protein is accompanied by a drastic change in the targeting of V(D)J recombinase activity, from being predominantly active at the heavy chain locus in pro-B cells to being exclusively restricted to the light chain loci in pre-B cells. This switch in locus-specific recombinase activity results in allelic exclusion at the immunoglobulin heavy chain locus. Allelic exclusion is maintained by a different mechanism at the light chain locus. We find that immature, but not mature, B cells that already express a functional light chain protein can undergo continued light chain gene rearrangement, by replacement of the original rearrangement on the same allele. Finally, we find that the developmentally regulated targeting of V(D)J recombination is unaffected by enforced rapid transit through the cell cycle induced by an Eµ-myc transgene.

A. Constantinescu was supported by grant no. GM07309 from the National Scientist Training Program. M.S. Schlissel is a Culpeper Scholar and a Leukemia Society Scholar. This work was supported by National Institutes of Health grant No. RO1 HL48702.

¹ Abbreviations used in this paper: 7-AAD, 7-aminoactinomycin D; CBE, coding broken ends; IgH, immunoglobulin heavy chain; Igµ, µ isotype; LMPCR, ligation-mediated PCR; pre-BCR, pre–B cell receptor; RAG, recombinase activating gene; RSS, recombination signal sequence; SBE, signal broken ends; SLC, surrogate light chain.

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