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 Cmu to a downstream Ch (for example, Cgamma, Cepsilon or Calpha), 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. For CSR, double-strand breaks are introduced into switch regions that flank Cmu and a downstream Ch, followed by fusion of the broken switch regions. 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. 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. CSR joins also display direct and microhomology joins, and CSR has been suggested to use C-NHEJ. 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-NHEJ. 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.

In mammalian cells, DNA double-strand breaks (DSBs) cause rapid phosphorylation of the H2AX core histone variant (to form ␥-H2AX) in megabase chromatin domains flanking sites of DNA damage. To investigate the role of H2AX in mammalian cells, we generated H2AX-deficient (H2AX ⌬/⌬ ) mouse embryonic stem (ES) cells. H2AX ⌬/⌬ ES cells are viable. However, they are highly sensitive to ionizing radiation (IR) and exhibit elevated levels of spontaneous and IR-induced genomic instability. Notably, H2AX is not required for NHEJ per se because H2AX ⌬/⌬ ES cells support normal levels and fidelity of V(D)J recombination in transient assays and also support lymphocyte development in vivo. However, H2AX ⌬/⌬ ES cells exhibit altered IR-induced BRCA1 focus formation. Our findings indicate that H2AX function is essential for mammalian DNA repair and genomic stability. The DNA in eukaryotic cells is packaged into chromatin, the fundamental unit of which is the nucleosome. The nucleosome consists of DNA wrapped around an octamer of the four core histones-H2A, H2B, H3, and H4 (1). The H2A histones consist of several subfamilies that contain distinct, conserved amino-and carboxyl-terminal amino acid sequences (2). The H2AX subfamily contains a conserved carboxyl-terminal SerGln-Glu (SQE motif) amino acid sequence. This SQE motif represents the consensus in vitro phosphorylation site for members of the phosphoinositide 3-kinase related kinase (PIKK) family that includes the protein kinases DNA-dependent protein kinase catalytic subunit (DNA-PKcs), ataxia telangiectasia mutated (ATM), and ATM and Rad3-related (ATR) (3).The repair of spontaneous and induced DNA double-strand breaks (DSBs) is critical for the maintenance of genomic integrity. In eukaryotic cells, the two major pathways of DSB repair are nonhomologous end-joining (NHEJ) and homologous recombination (HR; refs. 4 and 5). Covalent modifications of core histones via phosphorylation, acetylation, and methylation have been proposed to form a ''histone code'' that is read by cellular proteins to facilitate downstream molecular events (6). In response to DNA damage by agents that induce DNA doublestrand breaks, Mec1, the Saccharomyces cerevisiae homologue of ATR, phosphorylates the SQE motif of H2A (7). This phosphorylation event is required for the efficient repair of chromosomal DSBs by NHEJ but does not appear to be as important for homologous recombination (7). In mammalian cells, H2AX is rapidly phosphorylated on the induction of DSBs by ionizing radiation (IR) and DNA damaging agents (8, 9), resulting in formation of ␥-H2AX foci along megabase chromatin domains flanking DNA damage sites (9).Foci of ␥-H2AX also form at the immunoglobulin heavy chain locus during class switch recombination (CSR) in activated mature B cells (10). CSR occurs between large, highly repetitive S regions and also may be initiated by DSBs (10, 11) and completed by NHEJ factors (12)(13)(14)(15). Notably, CSR is significantly impaired in the absence of H2AX (10). Earlier during lymphocyte development...