DNA ligase IV (LIG4) is a nonhomologous end-joining (NHEJ) protein used for V(D)J recombination and DNA repair. In mice, Lig4 deficiency causes embryonic lethality, massive neuronal apoptosis, arrested lymphogenesis, and various cellular defects. Herein, we assess potential roles in this phenotype for INK4a/ARF and p53, two proteins implicated in apoptosis and senescence. INK4a/ARF deficiency rescued proliferation/senescence defects of Lig4-deficient fibroblasts but not other phenotypic aspects. In contrast, p53 deficiency rescued embryonic lethality, neuronal apoptosis, and fibroblast proliferation/senescence defects but not lymphocyte development or radiosensitivity. Young Lig4/p53 double null mice routinely died from pro-B lymphomas. Thus, in the context of Lig4 deficiency, embryonic lethality and neuronal apoptosis likely result from a p53-dependent response to unrepaired DNA damage, and neuronal apoptosis and lymphocyte developmental defects can be mechanistically dissociated.

Type I topoisomerases alter DNA topology by cleaving and rejoining one strand of duplex DNA through a covalent protein-DNA intermediate. Here we show that vaccinia topoisomerase, a eukaryotic type IB enzyme, catalyzes site-specific endoribonucleolytic cleavage of an RNA-containing strand. The RNase reaction occurs via transesterification at the scissile ribonucleotide to form a covalent RNA-3'-phosphoryl-enzyme intermediate, which is then attacked by the vicinal 2' OH of the ribose sugar to yield a free 2', 3' cyclic phosphate product. Introduction of a single ribonucleoside at the scissile phosphate of an otherwise all-DNA substrate suffices to convert the topoisomerase into an endonuclease. Human topoisomerase I also has endoribonuclease activity. These findings suggest potential roles for topoisomerases in RNA processing.

Hypomorphic mutations in the genes encoding the MRE11/RAD50/NBS1 (MRN) DNA repair complex lead to cancer-prone syndromes. MRN binds DNA double strand breaks where it functions in repair and triggers cell cycle checkpoints via activation of the ataxia-telangiectasia mutated (ATM) kinase. To gain understanding of MRN in cancer, we engineered mice with B lymphocytes lacking MRN, or harboring MRN in which MRE11 lacks nuclease activities. Both forms of MRN deficiency led to hallmarks of cancer, including oncogenic translocations involving c-Myc and the immunoglobulin locus. These pre-neoplastic B lymphocytes did not progress to detectable B lineage lymphoma, even in the absence of p53. Moreover, Mre11 deficiencies prevented tumorigenesis in a mouse model strongly predisposed to spontaneous B cell lymphomas. Our findings indicate that MRN cannot be considered a standard tumor suppressor and instead imply that nuclease activities of MRE11 are required for oncogenesis. Inhibition of MRE11 nuclease activity increased DNA damage and selectively induced apoptosis in cells overexpressing oncogenes, suggesting MRE11 serves an important role in countering oncogene-induced replication stress. Thus, MRE11 may offer a target for cancer therapeutic development. More broadly, our work supports the idea that subtle enhancements of endogenous genome instability can exceed the tolerance of cancer cells and be exploited for therapeutic ends.

No abstract

Allelic exclusion describes the essential immunological process by which feedback repression of sequential DNA rearrangements ensures that only one autosome expresses a functional T or B cell receptor. In wild-type mammals, approximately 60% of cells have recombined the DNA of one T cell receptor ␤ (TCR␤) V-to-DJ-joined allele in a functional configuration, while the second allele has recombined only the DJ sequences; the other 40% of cells have recombined the V to the DJ segments on both alleles, with only one of the two alleles predicting a functional TCR␤ protein. Here we report that the transgenic overexpression of GATA3 leads predominantly to biallelic TCR␤ gene (Tcrb) recombination. We also found that wild-type immature thymocytes can be separated into distinct populations based on intracellular GATA3 expression and that GATA3 LO cells had almost exclusively recombined only one Tcrb locus (that predicted a functional receptor sequence), while GATA3 HI cells had uniformly recombined both Tcrb alleles (one predicting a functional and the other predicting a nonfunctional rearrangement). These data show that GATA3 abundance regulates the recombination propensity at the Tcrb locus and provide new mechanistic insight into the historic immunological conundrum for how Tcrb allelic exclusion is mediated.KEYWORDS allelic exclusion, T cell receptor beta locus, GATA3, monoallelic-tobiallelic switch O ne enduring mystery in cellular immunology regards the underlying mechanisms that control antigen receptor allelic exclusion (1, 2), the process whereby B or T lymphocytes are programmed to express only one functional allele for each chain of their respective antigen receptors (B cell receptor [BCR] or T cell receptor [TCR]), thus avoiding the coexistence of multiple antigen specificities in a single immune cell. Lymphocytes acquire the diversity of antigen recognition (3) as well as a unique monospecificity for particular antigens (4) during development in the bone marrow (B cells) or the thymus (T cells).T lymphocyte development is generally characterized by division into multiple stages based on developmental timing and the location and expression of specific cell surface markers (5). T cell development begins when multipotential hematopoietic progenitor cells in the bone marrow migrate through the bloodstream to the thymus, where early T lineage progenitors (ETPs) are generated and later specified to become T cells (6-10). ETPs differentiate into double-negative (DN) cells (DN2 to DN4 stages) that express neither the CD4 nor the CD8 coreceptor, then into double-positive (DP)

In addition to its fundamental role of nucleating the formation of stable transcription complexes, the Xenopus laevis 5S RNA specific transcription factor, TFIIIA, promotes a variety of DNA-associated metabolic reactions. We report that TFIIIA can induce a DNA supercoiling catalyzed by the Xenopus laevis S-150 cell-free extract on plasmids containing a single copy of the Xenopus 5S RNA gene (somatic-type). Stimulated supercoiling occurs in the presence of high concentrations of ATP (4 mM) and at a factor to DNA ratio of 1 through a mechanism most likely involving type I topoisomerase. The highest level of stimulated supercoiling occurs when TFIIIA is incubated with DNA prior to the addition of the S-150 extract. Taken together, the experiments outlined in this report establish a reliable and seminal system in which TFIIIA-induced DNA supercoiling can be observed reproducibly.