1. Howard Hughes Medical Institute,
2. The Children's Hospital,
3. Immune Disease Institute,
4. Department of Genetics,
5. Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115, USA

Correspondence to: Frederick W. Alt^1,2,3,4 Correspondence and requests for materials should be addressed to F.W.A. (Email: alt@enders.tch.harvard.edu).

Abstract

Immunoglobulin variable region exons are assembled in developing B cells by V(D)J recombination. Once mature, these cells undergo class-switch recombination (CSR) when activated by antigen. CSR changes the heavy chain constant region exons (Ch) expressed with a given variable region exon from C to a downstream Ch (for example, C, C or C), thereby switching expression from IgM to IgG, IgE or IgA. Both V(D)J recombination and CSR involve the introduction of DNA double-strand breaks and their repair by means of end joining^{1, 2}. For CSR, double-strand breaks are introduced into switch regions that flank C and a downstream Ch, followed by fusion of the broken switch regions¹. In mammalian cells, the 'classical' non-homologous end joining (C-NHEJ) pathway repairs both general DNA double-strand breaks and programmed double-strand breaks generated by V(D)J recombination^{2, 3}. C-NHEJ, as observed during V(D)J recombination, joins ends that lack homology to form 'direct' joins, and also joins ends with several base-pair homologies to form microhomology joins^{3, 4}. CSR joins also display direct and microhomology joins, and CSR has been suggested to use C-NHEJ^{5, 6, 7, 8}. Xrcc4 and DNA ligase IV (Lig4), which cooperatively catalyse the ligation step of C-NHEJ, are the most specific C-NHEJ factors; they are absolutely required for V(D)J recombination and have no known functions other than C-NHEJ². Here we assess whether C-NHEJ is also critical for CSR by assaying CSR in Xrcc4- or Lig4-deficient mouse B cells. C-NHEJ indeed catalyses CSR joins, because C-NHEJ-deficient B cells had decreased CSR and substantial levels of IgH locus (immunoglobulin heavy chain, encoded by Igh) chromosomal breaks. However, an alternative end-joining pathway, which is markedly biased towards microhomology joins, supports CSR at unexpectedly robust levels in C-NHEJ-deficient B cells. In the absence of C-NHEJ, this alternative end-joining pathway also frequently joins Igh locus breaks to other chromosomes to generate translocations.

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