FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2024144876> ?p ?o ?g. }

• W2024144876 endingPage "13860" @default.
• W2024144876 startingPage "13857" @default.
• W2024144876 abstract "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·DNA-ligase IV complex. We show here that Cernunnos physically interacts with the XRCC4·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. 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·DNA-ligase IV complex. We show here that Cernunnos physically interacts with the XRCC4·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. 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 (1Revy P. Buck D. Le Deist F. de Villartay J.P. Adv. Immunol. 2005; 87: 237-295Crossref PubMed Scopus (56) Google Scholar). 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) 6The abbreviations used are: NHEJ, nonhomologous end joining; HCA, hydrophobic cluster analysis. 6The abbreviations used are: NHEJ, nonhomologous end joining; HCA, hydrophobic cluster analysis. pathways (see Ref. 2Sancar A. Lindsey-Boltz L.A. Unsal-Kacmaz K. Linn S. Annu. Rev. Biochem. 2004; 73: 39-85Crossref PubMed Scopus (2492) Google Scholar for review). Six mammalian factors constitute the core NHEJ apparatus: the Ku70/ Ku80 heterodimer, the DNA-PKcs kinase, the Artemis endonuclease, and the XRCC4·DNA-ligase IV complex responsible for the final ligation step (3Weterings E. van Gent D.C. DNA Repair (Amst.). 2004; 3: 1425-1435Crossref PubMed Scopus (168) Google Scholar). Additional NHEJ factors have been recognized in yeast, such as Nej1, which interacts with the yeast XRCC4 homolog Lif1p (4Valencia M. Bentele M. Vaze M.B. Herrmann G. Kraus E. Lee S.E. Schar P. Haber J.E. Nature. 2001; 414: 666-669Crossref PubMed Scopus (182) Google Scholar, 5Frank-Vaillant M. Marcand S. Genes Dev. 2001; 15: 3005-3012Crossref PubMed Scopus (134) Google Scholar, 6Kegel A. Sjostrand J.O. Astrom S.U. Curr. Biol. 2001; 11: 1611-1617Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). We recently identified a novel human NHEJ DNA repair factor, Cernunnos, the defect of which results in immune deficiency and microcephaly (7Buck D. Malivert L. de Chasseval R. Barraud A. Fondanèche M.C. Sanal O. Plebani A. Stéphan J.L. Hufnagel M. Deist F. Le Fischer A. Durandy A. de Villartay J.P. Revy P. Cell. 2006; 124: 287-299Abstract Full Text Full Text PDF PubMed Scopus (579) Google Scholar). One striking characteristic of Cernunnos patients is their clinical and biological resemblance with DNA-ligase IV patients (8O'Driscoll M. Cerosaletti K.M. Girard P.M. Dai Y. Stumm M. Kysela B. Hirsch B. Gennery A. Palmer S.E. Seidel J. Gatti R.A. Varon R. Oettinger M.A. Neitzel H. Jeggo P.A. Concannon P. Mol. Cell. 2001; 8: 1175-1185Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar, 9Ben-Omran T.I. Cerosaletti K. Concannon P. Weitzman S. Nezarati M.M. Am. J. Med. Genet. A. 2005; 137: 283-287Crossref Scopus (86) Google Scholar, 10Buck D. Moshous D. de Chasseval R. Ma Y. Le Deist F. Cavazzana-Calvo M. Fischer A. Casanova J.L. Lieber M.R. De Villartay J.P. Eur. J. Immunol. 2006; 36: 224-235Crossref PubMed Scopus (155) Google Scholar). 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 XRCC4·DNA-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 (7Buck D. Malivert L. de Chasseval R. Barraud A. Fondanèche M.C. Sanal O. Plebani A. Stéphan J.L. Hufnagel M. Deist F. Le Fischer A. Durandy A. de Villartay J.P. Revy P. Cell. 2006; 124: 287-299Abstract Full Text Full Text PDF PubMed Scopus (579) Google Scholar) and used to transfect 293T cells. Cells were lysed for 20 min on ice in 1 ml of lysis buffer (1× 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 mouse IgG (Santa Cruz Biotechnology) and then incubated (1 h, 4 °C) with 20 μl of prewashed protein A-Sepharose beads (Amersham Biosciences). Immunoprecipitations were performed on precleared lysates using anti-V5 (Invitrogen), anti-DNA-ligase IV (Acris), and anti-XRCC4 (Serotec) rabbit polyclonal or murine monoclonal anti-myc (Santa Cruz Biotechnology) antibodies for 1 h at 4 °C. Immune complexes were collected with protein A-Sepharose beads. Immunoprecipitates were analyzed by Western blotting with murine monoclonal anti-V5 (Invitrogen), anti-myc, or rabbit polyclonal anti-DNA-ligase IV and anti-XRCC4 antibodies.In Silico Sequence Analysis: Molecular Modeling—PSI-BLAST (11Altschul S.F. Madden T.L. Schaffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (59206) Google Scholar) searches were performed at NCBI (www.ncbi.nlm.nih.gov/blast) using the nr data base (2,920,020 sequences) (BLAST2.2.12, inclusion threshold E-value = 0.005). Guidelines to the use of hydrophobic cluster analysis (HCA) are given in Refs. 12Gaboriaud C. Bissery V. Benchetrit T. Mornon J.P. FEBS Lett. 1987; 224: 149-155Crossref PubMed Scopus (541) Google Scholar and 13Callebaut I. Labesse G. Durand P. Poupon A. Canard L. Chomilier J. Henrissat B. Mornon J.P. Cell Mol. Life Sci. 1997; 53: 621-645Crossref PubMed Scopus (430) Google Scholar. Modeling of three-dimensional structure was done using Modeler (14Sali A. Blundell T.L. J. Mol. Biol. 1993; 234: 779-815Crossref PubMed Scopus (10395) Google Scholar).RESULTS AND DISCUSSIONCernunnos Interacts with the XRCC4·DNA-Ligase IV Complex—We searched for a possible interaction of Cernunnos with the XRCC4·DNA-Ligase IV complex by transfecting 293T cells with a V5 epitope-tagged form of Cernunnos followed by immunoprecipitation experiments. As shown in Fig. 1A, the immunoprecipitation of Cernunnos via the V5 tag co-precipitated DNA-ligase IV (lane 6, upper panel). Conversely, the immunoprecipitation of either endogenous DNA-ligase IV (lane 8) or XRCC4 (lane 10) co-precipitated the transfected Cernunnos protein (revealed by anti-V5 antibody, bottom panel) in both cases. The same co-immunoprecipitation results were obtained (Fig. 1B) using the myc epitope-tagged version of Cernunnos. This demonstrates that XRCC4, DNA-ligase IV, and Cernunnos are part of a same complex. While this manuscript was under review, Ahnesorg et al. (15Ahnesorg P. Smith P. Jackson S.P. Cell. 2006; 124: 301-313Abstract Full Text Full Text PDF PubMed Scopus (582) Google Scholar) reported on the identification of XLF, a factor identical to Cernunnos, through its physical interaction with XRCC4.Cernunnos Is Homologous to XRCC4—To get further insight into the role of Cernunnos in NHEJ, we performed careful bioinformatics analysis of its protein sequence. Using HCA, a sensitive two-dimensional approach that is well suited to analyze the two-dimensional texture of proteins and compare their secondary structures (12Gaboriaud C. Bissery V. Benchetrit T. Mornon J.P. FEBS Lett. 1987; 224: 149-155Crossref PubMed Scopus (541) Google Scholar, 13Callebaut I. Labesse G. Durand P. Poupon A. Canard L. Chomilier J. Henrissat B. Mornon J.P. Cell Mol. Life Sci. 1997; 53: 621-645Crossref PubMed Scopus (430) Google Scholar), we first identified in the Cernunnos sequence a globular domain, ranging from amino acids 1–230. Using this sequence as query in PSI-BLAST (11Altschul S.F. Madden T.L. Schaffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (59206) Google Scholar) searches with default parameters, we detected different homologs of Cernunnos at convergence by iteration 4. A few of these sequences are shown in Fig. 2A (yellow box). A marginal similarity with a putative DNA double strand break repair protein from Arabidopsis thaliana was observed just above the threshold E-value (E-value = 0.28; 16% of identity over 108 amino acids). This protein turned out to be the plant XRCC4 homolog (white box in Fig. 2A), sharing with the human protein 24% sequence identity over the head and stalk regions. The similarity reported by PSI-BLAST between human Cernunnos and Arabidopsis XRCC4 encompasses the globular “head” domain of XRCC4 and the initial portion of the helical stalk, the overall region that promotes XRCC4 dimerization (16Junop M.S. Modesti M. Guarne A. Ghirlando R. Gellert M. Yang W. EMBO J. 2000; 19: 5962-5970Crossref PubMed Scopus (149) Google Scholar, 17Lee K.J. Huang J. Takeda Y. Dynan W.S. J. Biol. Chem. 2000; 275: 34787-34796Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 18Modesti M. Junop M.S. Ghirlando R. van de Rakt M. Gellert M. Yang W. Kanaar R. J. Mol. Biol. 2003; 334: 215-228Crossref PubMed Scopus (67) Google Scholar, 19Modesti M. Hesse J.E. Gellert M. EMBO J. 1999; 18: 2008-2018Crossref PubMed Scopus (151) Google Scholar, 20Sibanda B.L. Critchlow S.E. Begun J. Pei X.Y. Jackson S.P. Blundell T.L. Pellegrini L. Nat. Struct. Biol. 2001; 8: 1015-1019Crossref PubMed Scopus (211) Google Scholar). The similarity was supported at the two-dimensional level using HCA, revealing the conservation of the secondary structures forming the head domain, which contains a β-sandwich and two α-helices, as well as the helical stalk (Figs. 2A and 3A and data not shown). Most of the conserved features correspond to hydrophobicity (positions in which hydrophobicity is conserved are green in Fig. 2, with highly conserved aromatic residues in purple). These amino acids mainly participate in the head core structure or are involved in the contact with the initial portion of the stalk (e.g. Trp13 (W13) in Fig. 3A). Only a few surface-exposed residues are conserved, such as Phe117 and Trp119, which may be part of a potential interaction site (Fig. 3A). The finding of deleterious missense mutations (R57G and C123R in Fig. 3A) in the vicinity of these residues in patients (7Buck D. Malivert L. de Chasseval R. Barraud A. Fondanèche M.C. Sanal O. Plebani A. Stéphan J.L. Hufnagel M. Deist F. Le Fischer A. Durandy A. de Villartay J.P. Revy P. Cell. 2006; 124: 287-299Abstract Full Text Full Text PDF PubMed Scopus (579) Google Scholar) further strengthens the likely functional significance of this region of Cernunnos. Other PSI-BLAST searches were performed using Cernunnos homologous sequences as queries, which highlighted marginal similarities with additional XRCC4 proteins (E-value = >0.001), also supported at the two-dimensional level (data not shown). This first finding thus reveals a novel family of structurally similar proteins, gathering XRCC4 and Cernunnos. When our manuscript was under review, a similar conclusion was drawn by Ahnesorg et al. (15Ahnesorg P. Smith P. Jackson S.P. Cell. 2006; 124: 301-313Abstract Full Text Full Text PDF PubMed Scopus (582) Google Scholar), who used fold recognition methods to unravel the structural relationship between Cernunnos/XLF and XRCC4. However, these automatic methods proposed two different alignments of Cernunnos/XLF with the two available experimental structures of XRCC4, which yet share an almost identical sequence (99% identity; see Fig. 1 of supplemental data in Ref. 15Ahnesorg P. Smith P. Jackson S.P. Cell. 2006; 124: 301-313Abstract Full Text Full Text PDF PubMed Scopus (582) Google Scholar). Here, we propose a refined alignment of the two protein sequences, performed using the sensitive HCA methodology. This one benefited from the additional evolutionary information coming from the direct consideration of the whole Cernunnos and XRCC4 families. Accordingly, the alignment of the Cernunnos family sequences well conserved the structural invariants constituting the XRCC4 hydrophobic core (positions are shaded green and violet in Fig. 2A). These features were reinforced when considering the Nej1 sequences (see below).FIGURE 2Multiple sequence alignments of members of the Cernunnos/Nej1 family and XRCC4 proteins. A, the “head.” The alignment was constructed on the basis of PSI-BLAST results and manually refined using HCA. The D. hansenii protein has been significantly detected as a Cernunnos homolog as well as a Nej1 homolog (horizontal green box at the intersection of the yellow (Cernunnos) and blue (Nej1) boxes). It thus constitutes the intermediate protein, which allows revealing the relationship between Nej1 and Cernunnos. There is 15.8% sequence identity between the sequences of human Cernunnos and D. hansenii Cernunnos/Nej1 (119 aligned positions) and 10.3% between the sequences of D. hansenii Cernunnos/Nej1 and S. cerevisiae Nej1 (116 aligned positions). Secondary structures, as observed in the three-dimensional structures of XRCC4 (Protein Data Bank identifier 1FU1) are indicated above the XRCC4 sequences. Coloring scheme: white letters on a green background, positions in which hydrophobicity is conserved (on a purple background, conserved aromatic residues (FWY)); black letters on a gray background, other noticeable conserved positions. A, C, and T, which can substitute for hydrophobic residues (VILFMYW), are reported accordingly. GenBank™ identifiers (gi) are: 12084562 (Homo sapiens XRCC4), 9800643 (A. thaliana XRCC4), 50415440 (Xenopus laevis Cernunnos), 47550775 (Danio rerio Cernunnos), 40741672 (A. nidulans Cernunnos), 7160259 (S. pombe Cernunnos), 49657009 (D. hansenii Cernunnos/Nej1), 46442216 (C. albicans Nej1), 49645129 (K. lactis Nej1), and 44986293 (A. gossypii Nej1). Cernunnos sequence is from EMBL AJ972687. B, the DNA-ligase IV interaction region. The alignment gathers sequences of XRCC4 and Cernunnos from several species. XRCC4, but not Cernunnos, is found in plants (A. thaliana). The conserved sequences found in a similar location within the stalk in yeast Cernunnos/Nej1 proteins is not shown, as the corresponding profile poorly matches those of metazoan sequences. GenBank™ identifiers (gi) are as the same as in B and also 50761983 (Gallus gallus XRCC4), 49257218 (X. laevis XRCC4), 41387194 (D. rerio XRCC4), and 50750592 (G. gallus Cernunnos).View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 3Model for the Cernunnos three-dimensional structure. A, model of the dimeric three-dimensional structure of the human Cernunnos head domain and the initial portion of the stalk (up to residue Gly141). The model was built on the basis of the alignment shown in Fig. 2, using the nearly symmetric dimer of XRCC4 as a template (Protein Data Bank identifier 1FU1 (16Junop M.S. Modesti M. Guarne A. Ghirlando R. Gellert M. Yang W. EMBO J. 2000; 19: 5962-5970Crossref PubMed Scopus (149) Google Scholar)). Secondary structures are colored as described in the legend to Fig. 2. The white/brown helices (modeled on the XRCC4 stalk, sequence alignment not shown) recall the overall architecture of the XRCC4 protein dimer (stalk). B, putative combinatorial association within the XRCC4·DNA-ligase IV·Cernunnos complex. XRCC4 protein (orange) has been described as dimer in association with DNA-ligase IV (blue) or tetramers (16Junop M.S. Modesti M. Guarne A. Ghirlando R. Gellert M. Yang W. EMBO J. 2000; 19: 5962-5970Crossref PubMed Scopus (149) Google Scholar, 17Lee K.J. Huang J. Takeda Y. Dynan W.S. J. Biol. Chem. 2000; 275: 34787-34796Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 18Modesti M. Junop M.S. Ghirlando R. van de Rakt M. Gellert M. Yang W. Kanaar R. J. Mol. Biol. 2003; 334: 215-228Crossref PubMed Scopus (67) Google Scholar, 19Modesti M. Hesse J.E. Gellert M. EMBO J. 1999; 18: 2008-2018Crossref PubMed Scopus (151) Google Scholar, 20Sibanda B.L. Critchlow S.E. Begun J. Pei X.Y. Jackson S.P. Blundell T.L. Pellegrini L. Nat. Struct. Biol. 2001; 8: 1015-1019Crossref PubMed Scopus (211) Google Scholar). Several combinatorial associations with Cernunnos (green) can be hypothesized.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Several reports, including structural studies, have proposed that XRCC4 forms dimers in association with DNA-ligase IV or tetramers, the DNA-ligase IV-interacting region, and the tetramer stabilization region overlapping in the helical tail of XRCC4 (Fig. 2B) (16Junop M.S. Modesti M. Guarne A. Ghirlando R. Gellert M. Yang W. EMBO J. 2000; 19: 5962-5970Crossref PubMed Scopus (149) Google Scholar, 17Lee K.J. Huang J. Takeda Y. Dynan W.S. J. Biol. Chem. 2000; 275: 34787-34796Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 18Modesti M. Junop M.S. Ghirlando R. van de Rakt M. Gellert M. Yang W. Kanaar R. J. Mol. Biol. 2003; 334: 215-228Crossref PubMed Scopus (67) Google Scholar, 19Modesti M. Hesse J.E. Gellert M. EMBO J. 1999; 18: 2008-2018Crossref PubMed Scopus (151) Google Scholar, 20Sibanda B.L. Critchlow S.E. Begun J. Pei X.Y. Jackson S.P. Blundell T.L. Pellegrini L. Nat. Struct. Biol. 2001; 8: 1015-1019Crossref PubMed Scopus (211) Google Scholar). Equilibrium sedimentation analysis associated with in vitro mutagenesis suggested that these two forms of the XRCC4 protein (dimers with DNA-ligase IV versus tetramers) are mutually exclusive (18Modesti M. Junop M.S. Ghirlando R. van de Rakt M. Gellert M. Yang W. Kanaar R. J. Mol. Biol. 2003; 334: 215-228Crossref PubMed Scopus (67) Google Scholar). Although more difficult to align in the helical tail, Cernunnos and XRCC4 share similarities, especially in the DNA-ligase IV binding region of XRCC4 (Fig. 2B). Lys188 (Cernunnos Lys207), one of the residues involved in a salt bridge with the DNA-ligase IV BRCA1 C terminus domain linker, appears conserved in several sequences, as well as the F180IXXL cluster (Cernunnos F199LXXF), which makes hydrophobic contacts with DNA-ligase IV (20Sibanda B.L. Critchlow S.E. Begun J. Pei X.Y. Jackson S.P. Blundell T.L. Pellegrini L. Nat. Struct. Biol. 2001; 8: 1015-1019Crossref PubMed Scopus (211) Google Scholar).The finding that Cernunnos is related to XRCC4 on the one hand and the demonstration that Cernunnos interacts with the XRCC4·DNA-ligase IV complex on the other hand raises the interesting issue of the possible combinatorial associations of these two proteins within XRCC4/Cernunnos dimers and/or tetramers (Fig. 3B) and the possible functional consequences of these various combinations. As a first way to address this issue, we performed co-immunoprecipitation experiments following cotransfection of 293T cells with V5 epitope-tagged Cernunnos and myc epitope-tagged Cernunnos. As shown in Fig. 1C, immunoprecipitation with anti-V5 and anti-myc co-precipitated the Cernunnos-myc and Cernunnos-V5, respectively. This suggests that Cernunnos, as described for XRCC4, can form homoduplexes or at least that two Cernunnos molecules are part of the same complex as also demonstrated by Ahnesorg et al. (15Ahnesorg P. Smith P. Jackson S.P. Cell. 2006; 124: 301-313Abstract Full Text Full Text PDF PubMed Scopus (582) Google Scholar). Although the definite answers to these questions need further in vitro and in vivo investigations, one can already postulate that, given the embryonic lethality of XRCC4 mutant mice (21Gao Y. Sun Y. Frank K.M. Dikkes P. Fujiwara Y. Seidl K.J. Sekiguchi J.M. Rathbun G.A. Swat W. Wang J. Bronson R.T. Malynn B.A. Bryans M. Zhu C. Chaudhuri J. Davidson L. Ferrini R. Stamato T. Orkin S.H. Greenberg M.E. Alt F.W. Cell. 1998; 95: 891-902Abstract Full Text Full Text PDF PubMed Scopus (573) Google Scholar) and the severe immunodeficiency and developmental anomalies of human Cernunnos-deficient patients (7Buck D. Malivert L. de Chasseval R. Barraud A. Fondanèche M.C. Sanal O. Plebani A. Stéphan J.L. Hufnagel M. Deist F. Le Fischer A. Durandy A. de Villartay J.P. Revy P. Cell. 2006; 124: 287-299Abstract Full Text Full Text PDF PubMed Scopus (579) Google Scholar), these two factors, although part of the same complex, are not redundant. Moreover, overexpression of XRCC4 in Cernunnos-deficient cells does not reverse the defective V(D)J recombination (supplemental Fig. S1) indicating that these two factors play specific parts during the NHEJ process.Cernunnos Is Homologous to Yeast Nej1—Several putative partners of XRCC4 and DNA-ligase IV have been identified in mammals and yeast. One of these, Nej1/Lif2p, has been identified in Saccharomyces cerevisiae through its interaction with Lif1p, the yeast homolog of XRCC4, and by virtue of the fact that it is transcriptionally repressed/expressed in yeast diploid and haploid cells respectively (4Valencia M. Bentele M. Vaze M.B. Herrmann G. Kraus E. Lee S.E. Schar P. Haber J.E. Nature. 2001; 414: 666-669Crossref PubMed Scopus (182) Google Scholar, 5Frank-Vaillant M. Marcand S. Genes Dev. 2001; 15: 3005-3012Crossref PubMed Scopus (134) Google Scholar, 6Kegel A. Sjostrand J.O. Astrom S.U. Curr. Biol. 2001; 11: 1611-1617Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 22Ooi S.L. Shoemaker D.D. Boeke J.D. Science. 2001; 294: 2552-2556Crossref PubMed Scopus (124) Google Scholar). Nej1 thus appears as a critical regulator of NHEJ activity in yeast cells undergoing mating type switching. Moreover, ectopic expression of Nej1 restores NHEJ in MATa/MATα diploid cells to some extent (4Valencia M. Bentele M. Vaze M.B. Herrmann G. Kraus E. Lee S.E. Schar P. Haber J.E. Nature. 2001; 414: 666-669Crossref PubMed Scopus (182) Google Scholar, 5Frank-Vaillant M. Marcand S. Genes Dev. 2001; 15: 3005-3012Crossref PubMed Scopus (134) Google Scholar, 6Kegel A. Sjostrand J.O. Astrom S.U. Curr. Biol. 2001; 11: 1611-1617Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). To date no homolog of Nej1 has been identified in other organisms, including the fission yeast Schizosaccharomyces pombe. Given their interaction with the XRCC4·DNA-ligase IV complex and their similar molecular weight, we tackled the idea that Cernunnos may represent the genuine mammalian Nej1 homolog.Cernunnos was identified from human to several yeast species (S. pombe, Neurospora crassa, Gibberella zeae, Magnaporthe grisea, Aspergillus nidulans, Debaryomyces hansenii, Yarrowia lipolytica) by data base mining (Fig. 2A, yellow and green boxes, and data not shown) but intriguingly could not be found within the PSI-BLAST significant results in the S. cerevisiae species. No candidate could be found within the non-significant alignments either. Taking the opposite approach, using the S. cerevisiae Nej1 sequence as query in a PSI-BLAST search (same parameters as above) highlighted marginal similarities with two yeast sequences (Kluyveromyces lactis (E-value = 0.038) and Aphis gossypii (E-value = 0.14), Fig. 2A, blue box), which could be further supported at the two-dimensional level (data not shown). To further broaden the analysis, these loose similarities were quoted with significant E-values and extended to other yeast species in a reciprocal strategy. For example starting from the A. gossypii sequence, we highlighted significant similarities at convergence by iteration 4 with the S. cerevisiae Nej1 (E-value = 2 × 10–29) but also with two other yeast species (Candida albicans (E-value = 8 × 10–62) and D. hansenii (E-value = 9 × 10–76) (“Nej1” sequences in Fig. 2, blue and green boxes). Surprisingly, the D. hansenii Nej1 sequence highlighted here (GenBank™ identifier 49657009) was identical to the D. hansenii Cernunnos sequence identified above, making this yeast species the critical intermediate sequence that reveals the Nej1/Cernunnos relationship (green box in Fig. 2). Indeed, no significant, or marginal, similarities could be directly detected between Nej1 and the human Cernunnos sequence.Altogether this analysis indicated that Cernunnos is one homolog of Nej1 and that both proteins belong to an extended XRCC4 family. The great variability of the Cernunnos/Nej1 sequences among species is reminiscent of that of the XRCC4 sequences, and we show that they are structurally related. Indeed, the yeast XRCC4 homolog, Lif1, shares very low sequence similarity with human XRCC4, which cannot be detected using PSI-BLAST. Regarding this observation, one can hypothesize that the Cernunnos/XRCC4 couple has evolved jointly and has led to similarly highly divergent sequences in human and yeast. This concerted evolution, together with the more pronounced conservation of the dimerization regions (the “head” regions) might reflect the physical interaction of the two proteins, which has been demonstrated for Nej1/Lif1.Conclusion—We demonstrate here that the newly identified DNA repair factor, Cernunnos, interacts with the XRCC4·DNA-ligase IV complex, is the mammalian homolog of the yeast Nej1 factor, and that both belong to an extended XRCC4 family. These findings have several important implications. As we discussed above, the similarity between XRCC4 and Cernunnos raises the question of the true nature of the XRCC4·Cernunnos·DNA-ligase IV complex. Do both proteins always participate in the same complex? What are the rules governing their combinatorial association? What is the exact specific role that both factors play during the catalysis of DNA end joining? Analyzing mutations identified in patients is often of great help to describe an important region of a protein but sometimes can be misleading in case of hypomorphic mutations. Indeed, although the 2BN cells appear to harbor the more drastic mutation (a frameshift at the fifth amino acid) (15Ahnesorg P. Smith P. Jackson S.P. Cell. 2006; 124: 301-313Abstract Full Text Full Text PDF PubMed Scopus (582) Google Scholar), the clinical presentation appears somehow less severe than the other described Cernunnos mutants (7Buck D. Malivert L. de Chasseval R. Barraud A. Fondanèche M.C. Sanal O. Plebani A. Stéphan J.L. Hufnagel M. Deist F. Le Fischer A. Durandy A. de Villartay J.P. Revy P. Cell. 2006; 124: 287-299Abstract Full Text Full Text PDF PubMed Scopus (579) Google Scholar, 23Dai Y. Kysela B. Hanakahi L.A. Manolis K. Riballo E. Stumm M. Harville T.O. West S.C. Oettinger M.A. Jeggo P.A. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 2462-2467Crossref PubMed Scopus (154) Google Scholar). In particular, the 2BN patient did not present with microcephaly. This strongly suggests that 2BN harbors a hypomorphic Cernunnos mutation through the translation reinitiation on a downstream methionine as proposed (15Ahnesorg P. Smith P. Jackson S.P. Cell. 2006; 124: 301-313Abstract Full Text Full Text PDF PubMed Scopus (582) Google Scholar). A very similar situation has been described in the case of a particular mutation in the Rag1 gene, which in theory should lead to a T-B-SCID condition but, instead, appears as leaky, leading to the development of lymphocytes to some extent (24Noordzij J.G. Verkaik N.S. Hartwig N.G. de Groot R. van Gent D.C. van Dongen J.J. Blood. 2000; 96: 203-209Crossref PubMed Google Scholar, 25Corneo B. Moshous D. Gungor T. Wulffrat N. Philippet P. Le Deist F. Fischer A. de Villartay J.P. Blood. 2001; 97: 2772-2776Crossref PubMed Scopus (180) Google Scholar).The likely high interaction potential of the head regions of Cernunnos and Nej1 with yet uncharacterized molecular partners is revealed by the prominent level of strong hydrophobic residues that these sequences contain (37, 38, and 42% for human Cernunnos, D. hansenii Cernunnos/Nej1, and S. cerevisiae Nej1, respectively). These levels are significantly higher than those currently observed for globular domains of proteins (26George R.A. Lin K. Heringa J. Nucleic Acids Res. 2005; 33: W160-W163Crossref PubMed Scopus (27) Google Scholar) 7I. Callebaut and J.-P. Mornon, unpublished observations. and suggest that several strong" @default.
• W2024144876 created "2016-06-24" @default.
• W2024144876 creator A5021119090 @default.
• W2024144876 creator A5029004287 @default.
• W2024144876 creator A5031904282 @default.
• W2024144876 creator A5071218093 @default.
• W2024144876 creator A5083064307 @default.
• W2024144876 creator A5086067101 @default.
• W2024144876 date "2006-05-01" @default.
• W2024144876 modified "2023-10-09" @default.
• W2024144876 title "Cernunnos Interacts with the XRCC4·DNA-ligase IV Complex and Is Homologous to the Yeast Nonhomologous End-joining Factor Nej1" @default.
• W2024144876 cites W1540495975 @default.
• W2024144876 cites W1667638673 @default.
• W2024144876 cites W1861478519 @default.
• W2024144876 cites W1895234828 @default.
• W2024144876 cites W1971003485 @default.
• W2024144876 cites W1972070781 @default.
• W2024144876 cites W1972961211 @default.
• W2024144876 cites W2000988406 @default.
• W2024144876 cites W2001784394 @default.
• W2024144876 cites W2007710180 @default.
• W2024144876 cites W2008411383 @default.
• W2024144876 cites W2008686043 @default.
• W2024144876 cites W2029896618 @default.
• W2024144876 cites W2041731996 @default.
• W2024144876 cites W2058336786 @default.
• W2024144876 cites W2059678690 @default.
• W2024144876 cites W2060001445 @default.
• W2024144876 cites W2065283382 @default.
• W2024144876 cites W2071206740 @default.
• W2024144876 cites W2084919692 @default.
• W2024144876 cites W2094917306 @default.
• W2024144876 cites W2102752109 @default.
• W2024144876 cites W2109456034 @default.
• W2024144876 cites W2116718924 @default.
• W2024144876 cites W2118399795 @default.
• W2024144876 cites W2134968854 @default.
• W2024144876 cites W2147923983 @default.
• W2024144876 cites W2149156322 @default.
• W2024144876 cites W2158714788 @default.
• W2024144876 doi "https://doi.org/10.1074/jbc.c500473200" @default.
• W2024144876 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/16571728" @default.
• W2024144876 hasPublicationYear "2006" @default.
• W2024144876 type Work @default.
• W2024144876 sameAs 2024144876 @default.
• W2024144876 citedByCount "121" @default.
• W2024144876 countsByYear W20241448762012 @default.
• W2024144876 countsByYear W20241448762013 @default.
• W2024144876 countsByYear W20241448762014 @default.
• W2024144876 countsByYear W20241448762015 @default.
• W2024144876 countsByYear W20241448762016 @default.
• W2024144876 countsByYear W20241448762017 @default.
• W2024144876 countsByYear W20241448762019 @default.
• W2024144876 countsByYear W20241448762020 @default.
• W2024144876 countsByYear W20241448762021 @default.
• W2024144876 countsByYear W20241448762022 @default.
• W2024144876 countsByYear W20241448762023 @default.
• W2024144876 crossrefType "journal-article" @default.
• W2024144876 hasAuthorship W2024144876A5021119090 @default.
• W2024144876 hasAuthorship W2024144876A5029004287 @default.
• W2024144876 hasAuthorship W2024144876A5031904282 @default.
• W2024144876 hasAuthorship W2024144876A5071218093 @default.
• W2024144876 hasAuthorship W2024144876A5083064307 @default.
• W2024144876 hasAuthorship W2024144876A5086067101 @default.
• W2024144876 hasBestOaLocation W20241448761 @default.
• W2024144876 hasConcept C102744134 @default.
• W2024144876 hasConcept C104317684 @default.
• W2024144876 hasConcept C122917832 @default.
• W2024144876 hasConcept C134935766 @default.
• W2024144876 hasConcept C145803527 @default.
• W2024144876 hasConcept C175114707 @default.
• W2024144876 hasConcept C185592680 @default.
• W2024144876 hasConcept C2779222958 @default.
• W2024144876 hasConcept C54355233 @default.
• W2024144876 hasConcept C552990157 @default.
• W2024144876 hasConcept C60748783 @default.
• W2024144876 hasConcept C64894306 @default.
• W2024144876 hasConcept C86803240 @default.
• W2024144876 hasConcept C95444343 @default.
• W2024144876 hasConceptScore W2024144876C102744134 @default.
• W2024144876 hasConceptScore W2024144876C104317684 @default.
• W2024144876 hasConceptScore W2024144876C122917832 @default.
• W2024144876 hasConceptScore W2024144876C134935766 @default.
• W2024144876 hasConceptScore W2024144876C145803527 @default.
• W2024144876 hasConceptScore W2024144876C175114707 @default.
• W2024144876 hasConceptScore W2024144876C185592680 @default.
• W2024144876 hasConceptScore W2024144876C2779222958 @default.
• W2024144876 hasConceptScore W2024144876C54355233 @default.
• W2024144876 hasConceptScore W2024144876C552990157 @default.
• W2024144876 hasConceptScore W2024144876C60748783 @default.
• W2024144876 hasConceptScore W2024144876C64894306 @default.
• W2024144876 hasConceptScore W2024144876C86803240 @default.
• W2024144876 hasConceptScore W2024144876C95444343 @default.
• W2024144876 hasIssue "20" @default.
• W2024144876 hasLocation W20241448761 @default.
• W2024144876 hasLocation W20241448762 @default.
• W2024144876 hasLocation W20241448763 @default.
• W2024144876 hasLocation W20241448764 @default.

• next