The mechanism by which the cytidine deaminase activation-induced deaminase (AID) acts at immunoglobulin heavy-chain class switch regions during mammalian class switch recombination (CSR) remains unclear. R-loops have been proposed as a basis for this targeting. Here, we show that the difference between various forms of the S locus that can or cannot undergo CSR correlates well with the locations and detectability of R-loops. The S R-loops can initiate hundreds of base pairs upstream of the core repeat switch regions, and the area where the R-loops initiate corresponds to the zone where the AID mutation frequency begins to rise, despite a constant density of WRC sites in this region. The frequency of R-loops is 1 in 25 alleles, regardless of the presence of the core S repeats, again consistent with the initiation of most R-loops upstream of the core repeats. These findings explain the surprisingly high levels of residual CSR in B cells from mice lacking the core S repeats but the marked reduction in CSR in mice with deletions of the region upstream of the core S repeats. These studies also provide the first analysis of how R-loop formation in the eukaryotic chromosome depends on the DNA sequence.Mammalian immunoglobulin (Ig) genes undergo two types of DNA recombination, in addition to a somatic hypermutation (SHM) process. V(D)J recombination occurs in early lymphocytes and assembles the variable-domain exon so that IgM can be made. Class switch recombination (CSR) occurs only at the Ig heavy-chain locus and is responsible for the change in the heavy-chain isotype from IgM to IgG, IgA, or IgE; this process is also called the heavy-chain isotype switch (6,17,29). CSR occurs at repetitive DNA elements called switch regions, which vary in sequence and length. All of the switch regions are more than 1 kb in length and consist of unit repeats of 25 to 80 bp. All are located downstream of a sterile transcript promoter, which is necessary for CSR. All have a G-rich nontemplate strand, and all are rich in sites at which a cytidine deaminase called activation-induced deaminase (AID) prefers to act, namely, WRC sites (37). The regional nature of CSR, which gave rise to the term regionally specific recombination (15), contrasts with the vast majority of other physiologic recombination systems in biology, which are regarded as site specific. The special features of switch regions (such as being long and repetitive and located downstream of a promoter, having Grich nontemplate strands, and not having sequences conserved among the different switch regions or among vertebrates that carry out CSR) suggested that the mechanism would be unusual relative to other recombination processes in biology.Investigators at the Honjo laboratory discovered the key lymphoid-specific enzyme for both CSR and SHM, AID (22,23). AID is a 26-kDa protein which deaminates C in DNA (5, 25) but only when that DNA is single stranded (2,26,36,38). A key question for CSR and SHM concerns how the DNA becomes single stranded. Because transcription appears to...

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

DNA repair gene defects are found in virtually all human glioblastomas, but the genetic evidence for a direct role remains lacking. Here we demonstrate that combined inactivation of the XRCC4 non-homologous end-joining (NHEJ) DNA repair gene and p53 efficiently induces brain tumours with hallmark characteristics of human proneural/classical glioblastoma. The murine tumours exhibit PTEN loss of function instigated by reduced PTEN mRNA, and increased phosphorylated inactivation and stability as a consequence of aberrantly elevated CK2 provoked by p53 ablation and irrevocably deregulated by NHEJ inactivation. This results in DNA damage-resistant cytoplasmic PTEN and CK2 expression, and the attenuation of DNA repair genes. CK2 inhibition restores PTEN nuclear distribution and DNA repair activities and impairs tumour but not normal cell survival. These observations demonstrate that NHEJ contributes to p53-mediated glioblastoma suppression, and reveal a crucial role for PTEN in the early DNA damage signalling cascade, the inhibition of which promotes tumorigenicity and drug-resistant survival.

Antibody switching involves class switch recombination (CSR) events between switch (S) regions located upstream of heavy chain constant (C) genes. Mechanisms targeting CSR to S-regions are not clear. Deletion of Sμ tandem repeat (SμTR) sequences causes CSR to shift into downstream regions that do not undergo CSR in WT B-cells, including the Cμ-region. We now find that, in SμTR−/− B cells, Sμ chromatin histone modification patterns also shift downstream relative to WT and coincide with SμTR−/− CSR locations. Our results suggest that histone H3 acetylation and methylation are involved in accessibility of switch regions and that these modifications are not dependent on the underlying sequence, but may be controlled by the location of upstream promoter or regulatory elements. Our studies also show RNA polymerase II (RNAPII) loading increases in the Eμ/Iμ region in stimulated B cells; these increases are independent of SμTR sequences. Longer Sμ deletions have been reported to eliminate increases in RNAPII density, therefore we suggest that sequences between Iμ and Sμ (possibly the Iμ splicing region as well as G-tracts that are involved in stable RNA:DNA complex formation during transcription) might control the RNAPII density increases.