Class switch recombination (CSR) in B lymphocytes is initiated by introduction of multiple DNA double-strand breaks (DSBs) into switch (S) regions that flank immunoglobulin heavy chain ( IgH ) constant region exons. CSR is completed by joining a DSB in the donor Sμ to a DSB in a downstream acceptor S region (e.g., Sγ1) by end-joining. In normal cells, many CSR junctions are mediated by classical nonhomologous end-joining (C-NHEJ), which employs the Ku70/80 complex for DSB recognition and XRCC4/DNA ligase 4 for ligation. Alternative end-joining (A-EJ) mediates CSR, at reduced levels, in the absence of C-NHEJ, even in combined absence of Ku70 and ligase 4, demonstrating an A-EJ pathway totally distinct from C-NHEJ. Multiple DSBs are introduced into Sμ during CSR, with some being rejoined or joined to each other to generate internal switch deletions (ISDs). In addition, S-region DSBs can be joined to other chromosomes to generate translocations, the level of which is increased by absence of a single C-NHEJ component (e.g., XRCC4). We asked whether ISD and S-region translocations occur in the complete absence of C-NHEJ (e.g., in Ku70/ligase 4 double-deficient B cells). We found, unexpectedly, that B-cell activation for CSR generates substantial ISD in both Sμ and Sγ1 and that ISD in both is greatly increased by the absence of C-NHEJ. IgH chromosomal translocations to the c-myc oncogene also are augmented in the combined absence of Ku70 and ligase 4. We discuss the implications of these findings for A-EJ in normal and abnormal DSB repair.

Inactivation of the XRCC4 nonhomologous end-joining factor in the mouse germ line leads to embryonic lethality, in association with apoptosis of newly generated, postmitotic neurons. We now show that conditional inactivation of the XRCC4 in nestin-expressing neuronal progenitor cells, although leading to no obvious phenotype in a WT background, leads to early onset of neuronally differentiated medulloblastomas (MBs) in a p53-deficient background. A substantial proportion of the XRCC4͞p53-deficient MBs have high-level N-myc gene amplification, often intrachromosomally in the context of complex translocations or other alterations of chromosome 12, on which N-myc resides, or extrachromosomally within double minutes. In addition, most XRCC4͞ p53-deficient MBs harbor clonal translocations of chromosome 13, which frequently involve chromosome 6 as a partner. One copy of the patched gene (Ptc), which lies on chromosome 13, was deleted in all tested XRCC4͞p53-deficient MBs in the context of translocations or interstitial deletions. In addition, Cyclin D2, a chromosome 6 gene, was amplified in a subset of tumors. Notably, amplification of Myc-family or Cyclin D2 genes and deletion of Ptc also have been observed in human MBs. We therefore conclude that, in neuronal cells of mice, the nonhomologous end-joining pathway plays a critical role in suppressing genomic instability that, in a p53-deficient background, routinely contributes to genesis of MBs with recurrent chromosomal alterations.

DNA ligase IV (LIG4) is an essential component of the nonhomologous end-joining (NHEJ) repair pathway and plays a key role in V(D)J recombination. Hypomorphic LIG4 mutations in humans are associated with increased cellular radiosensitivity, microcephaly, facial dysmorphisms, growth retardation, developmental delay, and a variable degree of immunodeficiency. We have generated a knock-in mouse model with a homozygous Lig4 R278H mutation that corresponds to the first LIG4 mutation reported in humans. The phenotype of homozygous mutant mice Lig4 R278H/R278H (Lig4 R/R ) includes growth retardation, a decreased life span, a severe cellular sensitivity to ionizing radiation, and a very severe, but incomplete block in T and B cell development. Peripheral T lymphocytes show an activated and anergic phenotype, reduced viability, and a restricted repertoire, reminiscent of human leaky SCID. Genomic instability is associated with a high rate of thymic tumor development. Finally, Lig4 R/R mice spontaneously produce low-affinity antibodies that include autoreactive specificities, but are unable to mount high-affinity antibody responses. These findings highlight the importance of LIG4 in lymphocyte development and function, and in genomic stability maintenance, and provide a model for the complex phenotype of LIG4 syndrome in humans.genomic instability | immunodeficiency | lymphocytes | nonhomologous end joining | immune dysregulation N onhomologous end joining (NHEJ) is one of the two major DNA repair pathways in mammalian cells that protect the genome against DNA double-stranded breaks (DSBs) generated by genomic insults such as ionizing radiation (IR) or reactive oxygen species or that arise during V(D)J recombination (1) and Ig heavy chain (IGH) class switch recombination (CSR) (2). V(D)J recombination is the process by which developing T and B lymphocytes assemble their antigen receptor variable region exons. In this process, the recombinase activating gene (RAG)1 and RAG2 proteins introduce DSBs at recombination signal sequences (RSS) that flank coding variable (V), diversity (D), and joining (J) elements. This process generates hairpin-sealed coding ends and blunt phosphorylated signal ends. These DNA ends are recognized and resolved by proteins of the NHEJ pathway, with formation of coding joins and recombination signal (RS) joins, respectively. In particular, the KU70/KU80 heterodimer binds directly the DNA ends, allowing activation of the DNA-PK catalytic subunit (DNA-PKcs) (3) and phosphorylation of Artemis, which mediates opening of hairpin-sealed DNA coding ends (4,5