Structure and mechanism of action of the BRCA2 breast cancer tumor suppressor.

Mutations in BRCA2 increase susceptibility to breast, ovarian and prostate cancers. The product of human BRCA2, BRCA2 protein, has a key role in the repair of DNA double-strand breaks and interstrand cross-links by RAD51-mediated homologous recombination. Here, we present a biochemical and structural characterization of full-length (3,418 amino acid) BRCA2, alone and in complex with RAD51. We show that BRCA2 facilitates nucleation of RAD51 filaments at multiple sites on single-stranded DNA. Three-dimensional EM reconstructions revealed that BRCA2 exists as a dimer and that two oppositely oriented sets of RAD51 molecules bind the dimer. Single-stranded DNA binds along the long axis of BRCA2, such that only one set of RAD51 monomers can form a productive complex with DNA and establish filament formation. Our data define the molecular mechanism by which this tumor suppressor facilitates RAD51-mediated homologous-recombinational repair.

Cernunnos deficiency reduces thymocyte life span and alters the T cell repertoire in mice and humans.

Cernunnos is a DNA repair factor of the nonhomologous end-joining machinery. Its deficiency in humans causes radiosensitive severe combined immune deficiency (SCID) with microcephaly, characterized in part by a profound lymphopenia. In contrast to the human condition, the immune system of Cernunnos knockout (KO) mice is not overwhelmingly affected. In particular, Cernunnos is dispensable during V(D)J recombination in lymphoid cells. Nevertheless, the viability of thymocytes is reduced in Cernunnos KO mice, owing to the chronic activation of a P53-dependent DNA damage response. This translates into a qualitative alteration of the T cell repertoire to one in which the most distal Vα and Jα segments are missing. This results in the contraction of discrete T cell populations, such as invariant natural killer T (iNKT) and mucosa-associated invariant T (MAIT) cells, in both humans and mice.

Delineation of the Xrcc4-interacting region in the globular head domain of cernunnos/XLF.

In mammals, the majority of DNA double-strand breaks are processed by the nonhomologous end-joining (NHEJ) pathway, composed of seven factors: Ku70, Ku80, DNA-PKcs, Artemis, Xrcc4 (X4), DNA-ligase IV (L4), and Cernunnos/XLF. Cernunnos is part of the ligation complex, constituted by X4 and L4. To improve our knowledge on the structure and function of Cernunnos, we performed a systematic mutagenesis study on positions selected from an analysis of the recent three-dimensional structures of this factor. Ten of 27 screened mutants were nonfunctional in several DNA repair assays. Outside amino acids critical for the expression and stability of Cernunnos, we identified three amino acids (Arg(64), Leu(65), and Leu(115)) essential for the interaction with X4 and the proper function of Cernunnos. Docking the crystal structures of the two factors further validated this probable interaction surface of Cernunnos with X4.

The C-terminal domain of Cernunnos/XLF is dispensable for DNA repair in vivo.

The core nonhomologous end-joining DNA repair pathway is composed of seven factors: Ku70, Ku80, DNA-PKcs, Artemis, XRCC4 (X4), DNA ligase IV (L4), and Cernunnos/XLF (Cernunnos). Although Cernunnos and X4 are structurally related and participate in the same complex together with L4, they have distinct functions during DNA repair. L4 relies on X4 but not on Cernunnos for its stability, and L4 is required for optimal interaction of Cernunnos with X4. We demonstrate here, using in vitro-generated Cernunnos mutants and a series of functional assays in vivo, that the C-terminal region of Cernunnos is dispensable for its activity during DNA repair.

JAK2 stimulates homologous recombination and genetic instability: potential implication in the heterogeneity of myeloproliferative disorders.

The JAK2(V617F) mutation is frequently observed in classical myeloproliferative disorders, and disease progression is associated with a biallelic acquisition of the mutation occurring by mitotic recombination. In this study, we examined whether JAK2 activation could lead to increased homologous recombination (HR) and genetic instability. In a Ba/F3 cell line expressing the erythropoietin (EPO) receptor, mutant JAK2(V617F) and, to a lesser extent, wild-type (wt) JAK2 induced an increase in HR activity in the presence of EPO without modifying nonhomologous end-joining efficiency. Moreover, a marked augmentation in HR activity was found in CD34(+)-derived cells isolated from patients with polycythemia vera or primitive myelofibrosis compared with control samples. This increase was associated with a spontaneous RAD51 foci formation. As a result, sister chromatid exchange was 50% augmented in JAK2(V617F) Ba/F3 cells compared with JAK2wt cells. Moreover, JAK2 activation increased centrosome and ploidy abnormalities. Finally, in JAK2(V617F) Ba/F3 cells, we found a 100-fold and 10-fold increase in mutagenesis at the HPRT and Na/K ATPase loci, respectively. Together, this work highlights a new molecular mechanism for HR regulation mediated by JAK2 and more efficiently by JAK2(V617F). Our study might provide some keys to understand how a single mutation can give rise to different pathologies.

DNA repair and the immune system: From V(D)J recombination to aging lymphocytes.

B and T lymphocytes are exposed to various genotoxic stresses during their life, which originate from programmed molecular mechanisms during their development and maturation or are secondary to cellular metabolism during acute phases of cell proliferation and activation during immune responses. How lymphocytes handle these multiple genomic assault has become a focus of interest over the years, perhaps beginning with the identification of the murine scid model in the early 80s when it was recognized that DNA repair deficiencies had profound consequences on the immune system. In this respect, the immune system represents an ideal model to study DNA damage responses (DDR) and the survey of immune deficiency conditions in humans or the development of specific animal models provided many major contributions in our understanding of the various biochemical pathways at play during DDR in general. Although the role of DNA repair in the early phases of B and T cell development has been analyzed thoroughly, the role of these functions in various aspects of the mature immune system (homeostasis, immunological memory, ageing) is less well understood. Lastly, the analysis of DNA repair in the immune system has provided many insights in the more general understanding of cancer.

Interplay between Cernunnos-XLF and nonhomologous end-joining proteins at DNA ends in the cell.

Cernunnos-XLF is the most recently identified core component in the nonhomologous end-joining (NHEJ) pathway for the repair of DNA double strand breaks (DSBs) in mammals. It associates with the XRCC4/ligase IV ligation complex and stimulates its activity in a still unknown manner. NHEJ also requires the DNA-dependent protein kinase that contains a Ku70/Ku80 heterodimer and the DNA-dependent protein kinase catalytic subunit. To understand the interplay between Cernunnos-XLF and the other proteins implicated in the NHEJ process, we have analyzed the interactions of Cernunnos-XLF and NHEJ proteins in cells after treatment with DNA double strand-breaking agents by means of a detergent-based cellular fractionation protocol. We report that Cernunnos-XLF is corecruited with the core NHEJ components on chromatin damaged with DSBs in human cells and is phosphorylated by the DNA-dependent protein kinase catalytic subunit. Our data show a pivotal role for DNA ligase IV in the NHEJ ligation complex assembly and recruitment to DSBs because the association of Cernunnos-XLF with the XRCC4/ligase IV complex relies primarily on the DNA ligase IV component, and an intact XRCC4/ligase IV complex is necessary for Cernunnos-XLF mobilization to damaged chromatin. Conversely, a Cernunnos-XLF defect has no apparent impact on the XRCC4/ligase IV association and recruitment to the DSBs or on the stimulation of the DNA-dependent protein kinase on DNA ends.

Cernunnos-XLF, a recently identified non-homologous end-joining factor required for the development of the immune system.

PURPOSE OF REVIEW: The analysis of immune deficiency conditions in humans has recently allowed the identification of a novel factor, Cernunnos, involved in DNA repair and required during the development of the immune system. The present review will focus on the information and new questions provided by the discovery of Cernunnos. RECENT FINDINGS: The study of human immune deficiency conditions associated with defective DNA repair led to the recent identification of Cernunnos. Cernunnos is required for the ubiquitous DNA repair process mainly used in mammals, the non-homologous end-joining pathway. The analysis of Cernunnos defect conditions demonstrated the essential role of this novel factor during the development of the immune system. Cernunnos is homologous to the non-homologous end-joining factor, XRCC4, and is the genuine homologue of the yeast non-homologous end-joining factor, Nej1. These observations shed new light on the process underlying the last step of the non-homologous end-joining pathway, the DNA ends ligation. SUMMARY: The elucidation of the molecular bases of rare inherited immune deficiencies associated with DNA repair defects provide considerable insights into our understanding of fundamental mechanisms such as the non-homologous end-joining ubiquitous DNA repair pathway.

Cernunnos interacts with the XRCC4 x DNA-ligase IV complex and is homologous to the yeast nonhomologous end-joining factor Nej1.

DNA double strand breaks are considered as the most harmful DNA lesions and are repaired by either homologous recombination or nonhomologous end joining (NHEJ). A new NHEJ factor, Cernunnos, has been identified, the defect of which leads to a severe immunodeficiency condition associated with microcephaly and other developmental defects in humans. This presentation is reminiscent to that of DNA-ligase IV deficiency and suggests a possible interplay between Cernunnos and the XRCC4 x DNA-ligase IV complex. We show here that Cernunnos physically interacts with the XRCC4 x DNA-ligase IV complex. Moreover, a combination of sensitive methods of sequence analysis revealed that Cernunnos can be associated with the XRCC4 family of proteins and that it corresponds to the genuine homolog of the yeast Nej1 protein. Altogether these results shed new lights on the last step, the DNA religation, of the NHEJ pathway.

Cernunnos, a novel nonhomologous end-joining factor, is mutated in human immunodeficiency with microcephaly.

DNA double-strand breaks (DSBs) occur at random upon genotoxic stresses and represent obligatory intermediates during physiological DNA rearrangement events such as the V(D)J recombination in the immune system. DSBs, which are among the most toxic DNA lesions, are preferentially repaired by the nonhomologous end-joining (NHEJ) pathway in higher eukaryotes. Failure to properly repair DSBs results in genetic instability, developmental delay, and various forms of immunodeficiency. Here we describe five patients with growth retardation, microcephaly, and immunodeficiency characterized by a profound T+B lymphocytopenia. An increased cellular sensitivity to ionizing radiation, a defective V(D)J recombination, and an impaired DNA-end ligation process both in vivo and in vitro are indicative of a general DNA repair defect in these patients. All five patients carry mutations in the Cernunnos gene, which was identified through cDNA functional complementation cloning. Cernunnos/XLF represents a novel DNA repair factor essential for the NHEJ pathway.