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.

f 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 V␣ and J␣ 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.T he immune system is the site of intense genome dynamics, in particular during the development and maturation of B and T lymphocytes in bone marrow and the thymus, when antigen receptor genes are rearranged through V(D)J recombination prior to their expression. DNA damages are also likely to occur during the several phases of intense proliferation which accompany the development of B and T cells.V(D)J recombination is the prototypical example of the generation of a programmed DNA double-strand break (DNA-dsb) during lymphoid development through the activity of recombination activating genes 1 and 2 (Rag1/2) on immunoglobulin (Ig) and T cell receptor (TCR) genes (see the work of Helmink and Sleckman [1] for a recent review). The resulting DNA-dsb is resolved by the nonhomologous end-joining (NHEJ) DNA repair pathway, composed of seven core components (see the work of Lieber [2] for a recent review). The Cernunnos-Xrcc4-DNA ligase IV complex ultimately reseals the DNA-dsb. Cernunnos, also known as Xrcc4-like factor (XLF), was the last NHEJ factor that was independently identified, through a survey of RS-SCID patients (3) and a yeast two-hybrid screen with Xrcc4 as a bait (4). Cernunnos and Xrcc4 adopt the same overall three-dimensional crystal structure (5, 6) and, together with DNA ligase IV, are parts of the same complex (4, 7). Cernunnos stimulates the DNA-joining activity of the Xrcc4-DNA ligase IV complex (8, 9). V(D)J recombination constitutes a central checkpoint in the development of the immune system, as its defect leads to abortive B and T cell maturation in vivo, resulting in severe combined immune deficiency (SCID) (10), but its first recognized function was the generation of a diverse antigenic repertoire through the combinatorial association of variable, diversity, and joining segments that encode the variable domains of both Ig and TCRs (11). Numerous examples show that a reduced V(D)J recombinase activity affects the extent of antigenic diversity of immune receptors in mice and humans. The resulting immune deregulation may then lead to autoimmunity, increased susceptibility to infections, or the development of vario...

Mutations in BRCA2 increase susceptibility to breast, ovarian and prostate cancers. The product of human BRCA2, BRCA2 protein, plays a key role in the repair of DNA double strand breaks and interstrand crosslinks 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 electron microscopy 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.

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 XRCC4⅐DNA-ligase IV complex. We show here that Cernunnos physically interacts with the XRCC4⅐DNA-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.DNA double strand breaks are caused by exposure to ionizing radiation or chemical agents. They also result from physiological DNA rearrangements occurring during meiosis or, in vertebrate cells, during specialized recombination events underlying the development and maturation of the adaptive immune system (1). Two main mechanisms were developed in eukaryotic cells for efficient DNA double strand break repair: the accurate homologous recombination and the nonhomologous end-joining (NHEJ) 6 pathways (see Ref. 2 for review). Six mammalian factors constitute the core NHEJ apparatus: the Ku70/ Ku80 heterodimer, the DNA-PKcs kinase, the Artemis endonuclease, and the XRCC4⅐DNA-ligase IV complex responsible for the final ligation step (3). Additional NHEJ factors have been recognized in yeast, such as Nej1, which interacts with the yeast XRCC4 homolog Lif1p (4 -6). We recently identified a novel human NHEJ DNA repair factor, Cernunnos, the defect of which results in immune deficiency and microcephaly (7). One striking characteristic of Cernunnos patients is their clinical and biological resemblance with DNAligase IV patients (8 -10). This includes microcephaly and immune deficiency, mostly characterized by a severely impaired development of B and T lymphocytes. Both conditions are caused by a faulty NHEJ, which translates into increased cellular sensitivity to DNA-damaging agents and the impaired capacity to join double-stranded DNA ends in vitro and in vivo. This parallel prompted us to hypothesize that Cernunnos may be acting at the same level as the XRCC4⅐DNA-ligase IV complex during DNA repair and may indeed incorporates into this complex. EXPERIMENTAL PROCEDURESAntibodies and Immunoprecipitation-The Cernunnos Orf, together with a C terminus V5 or myc epitope tag, was cloned into the pCDNA3.1 vector (7) and used to transfect 293T cells. Cells were lysed for 20 min on ice in 1 ml of lysis buffer (1ϫ TNE) containing 50 mM Tris (pH 8.0), 2 mM EDTA, 0.5% Nonidet P-40, 1% phosphatase inhibitor cocktails (1 and 2, Sigma), and protease inhibitor (Roche Applied Science). One mg of cell lysate was first precleared with rabbit or mous...

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. (Blood. 2008;112:1402-1412) IntroductionMyeloproliferative disorders (MPDs) are clonal hematopoietic malignancies in which one or several myeloid lineages (ie, granulocytic, erythroid, and megakaryocytic) are abnormally amplified. This amplification is thought to result from the deregulation of hematopoietic stem cells with a downstream selective proliferation advantage in late myeloid differentiation. MPDs are classified by the World Health Organization into 3 categories: (1) chronic myeloid leukemia (CML); (2) classical MPD, including polycythemia vera (PV), essential thrombocythemia (ET), and primitive myelofibrosis (PMF); and (3) unclassified and rare MPDs comprising chronic neutrophilic leukemia (CNL), hypereosinophilic syndrome/chronic eosinophilic leukemia (HEL/CEL), and other unclassifiable myeloproliferation.MPDs frequently involve the deregulation of a tyrosine kinase because of the acquisition of a monogenetic abnormality in a hematopoietic stem cell, the archetype of which is BCR/ABL in CML. Recently, several groups have identified a recurrent acquired mutation in the tyrosine Janus kinase 2 (JAK2) gene in most cases of PV patients and in approximately half of ET and PMF patients. [1][2][3][4][5] JAK2 is a tyrosine kinase that becomes activated downstream of several cytokine receptors after ligand binding, such as erythropoietin (EPO). JAK2 activation leads to activation of many signaling pathways, including the MAPK, PI3K, and STATs pathways. The V617F point mutation lies in the autoinhibitory JH2 domain of JAK2, and in vitro studies have demonstrated that the mutation leads to JAK2 autophosphorylation and to the constitutive...

The immune system is the site of intense DNA damage/ modification, which occur during the development and maturation of B and T lymphocytes. V(D)J recombination is initiated by the Rag1 and Rag2 proteins and the formation of a DNA double-strand break (DNA dsb). This DNA lesion is repaired through the use of the nonhomologous end-joining (NHEJ) pathway, several factors of which have been identified through the survey of immunodeficient conditions in humans and mice. Upon antigenic recognition in secondary lymphoid organs, mature B cells further diversify their repertoire through class switch recombination (CSR). CSR is a region-specific rearrangement process triggered by the activation-induced cytidine deaminase factor and also proceeds through the introduction of DNA dsb. However, unlike V(D)J recombination, CSR does not rely strictly on NHEJ for the repair of the DNA lesion. Instead, CSR, but not V(D)J recombination, requires the major factors of the DNA damage response. V(D)J recombination and CSR thus represent an interesting paradigm to study the regulation among the various DNA repair pathways. Oncogene (2007) 26, 7780-7791; doi:10.1038/sj.onc.1210875 Keywords: V(D)J recombination; class switch recombination; SCID; Artemis; Cernunnos; NHEJ V(D)J recombination and CSR: two rearrangement processes that shape the immune system repertoire B and T lymphocytes respond to foreign pathogens through specialized antigenic receptors, the B-cell receptor and T-cell receptor, respectively. The required diversity of these receptors is ensured by the V(D)J recombination process (Figure 1) which assembles previously scattered Variable (V), Diversity (D) and Joining (J) encoding gene segments through a specialized somatic DNA rearrangement mechanism (Tonegawa, 1983). The reaction is initiated by the lymphoid-specific factors, Rag1 and Rag2, which recognize recombination signal sequences (RSS) that flank all V, D and J gene units and introduce a DNA dsb at the border of the RSS (see Dudley et al. (2005) for a review on V(D)J recombination). The resulting DNA double-strand break (DNA dsb) is resolved by the ubiquitous DNA repair machinery known as non-homologous end-joining (NHEJ). The convergent efforts of scientists in the immunology and DNA repair fields were decisive in defining the various actors and molecular mechanisms of this DNA repair pathway over the years as will be discussed below.The terminal maturation of B lymphocytes, which occurs in germinal centres of secondary lymphoid organs upon antigen recognition, is accompanied by two additional molecular processing of immunoglobulin genes that increase the efficiency of the humoral response: (1) the class switch recombination (CSR, Figure 2) exchanges the immunoglobulin (Ig) constant region (CH), thus modifying the function of the Ig without altering its antigenic specificity (Manis et al., 2002b) and (2) somatic hypermutations are introduced in the Ig variable domains, thus increasing their affinity for antigen (Papavasiliou and Schatz, 2002). These two events ar...

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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.