XRCC4 Protein

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Matthias Mann - One of the best experts on this subject based on the ideXlab platform.

  • activity of dna ligase iv stimulated by complex formation with XRCC4 Protein in mammalian cells
    Nature, 1997
    Co-Authors: Ulf Grawunder, Matthias Wilm, Xiantuo Wu, Peter Kulesza, Thomas E Wilson, Matthias Mann
    Abstract:

    Mutation of the XRCC4 gene in mammalian cells1,2 prevents the formation of the signal and coding joints in the V(D)J recombination reaction3, which is necessary for production of a functional immunoglobulin gene, and renders the cells highly sensitive to ionizing radiation4. However, XRCC4 shares no sequence homology with other Proteins, nor does it have a biochemical activity to indicate what its function might be2. Here we show that DNA ligase IV (ref. 5) co-immunoprecipitates with XRCC4 and that these two Proteins specifically interact with one another in a yeast two-hybrid system. Ligation of DNA double-strand breaks in a cell-free system by DNA ligase IV is increased fivefold by purified XRCC4 and seven- to eightfold when XRCC4 is co-expressed with DNA ligase IV. We conclude that the biological consequences of mutating XRCC4 are primarily due to the loss of its stimulatory effect on DNA ligase IV: the function of the XRCC4–DNA ligase IV complex may be to carry out the final steps of V(D)J recombination and joining of DNA ends.

Martin Gellert - One of the best experts on this subject based on the ideXlab platform.

  • Tetramerization and DNA ligase IV interaction of the DNA double-strand break repair Protein XRCC4 are mutually exclusive.
    Journal of Molecular Biology, 2003
    Co-Authors: Mauro Modesti, Martin Gellert, Murray S. Junop, Rodolfo Ghirlando, Wei Yang, Mandy W.m.m. Van De Rakt, Roland Kanaar
    Abstract:

    The XRCC4 Protein is of critical importance for the repair of broken chromosomal DNA by non-homologous end joining (NHEJ). The absence of XRCC4 abolishes chromosomal NHEJ almost completely. One reason for this severe phenotype is that XRCC4 binds and modulates the stability and activity of the NHEJ-specific ligase, DNA ligase IV. XRCC4 in solution is in equilibrium between the dimeric and tetrameric forms. Previous structural studies have shown that the interface between dimers is located in the same region as that implicated in DNA ligase IV interaction. With the use of equilibrium sedimentation analysis, we show here that only the XRCC4 dimer can associate with DNA ligase IV, forming a monodisperse complex of 2:1 stoichiometry in solution. In addition, physical analysis of XRCC4/DNA ligase IV complex formation, combined with mutational analysis of XRCC4, indicates that tetramerization and DNA ligase IV binding are mutually exclusive. We propose that the putative function of the XRCC4 tetramer is distinct from its DNA ligase IV-associated function.

  • Crystal structure of the XRCC4 DNA repair Protein and implications for end joining.
    The EMBO Journal, 2000
    Co-Authors: Murray S. Junop, Mauro Modesti, Martin Gellert, Alba Guarné, Rodolfo Ghirlando, Wei Yang
    Abstract:

    XRCC4 is essential for carrying out non‐homologous DNA end joining (NHEJ) in all eukaryotes and, in particular, V(D)J recombination in vertebrates. XRCC4 Protein forms a complex with DNA ligase IV that rejoins two DNA ends in the last step of V(D)J recombination and NHEJ to repair double strand breaks. XRCC4‐defective cells are extremely sensitive to ionizing radiation, and disruption of the XRCC4 gene results in embryonic lethality in mice. Here we report the crystal structure of a functional fragment of XRCC4 at 2.7 A resolution. XRCC4 Protein forms a strikingly elongated dumb‐bell‐like tetramer. Each of the N‐terminal globular head domains consists of a β‐sandwich and a potentially DNA‐binding helix– turn–helix motif. The C‐terminal stalk comprising a single α‐helix >120 A in length is partly incorporated into a four‐helix bundle in the XRCC4 tetramer and partly involved in interacting with ligase IV. The XRCC4 structure suggests a possible mode of coupling ligase IV association with DNA binding for effective ligation of DNA ends.

  • dna binding of XRCC4 Protein is associated with v d j recombination but not with stimulation of dna ligase iv activity
    The EMBO Journal, 1999
    Co-Authors: Mauro Modesti, Joanne E Hesse, Martin Gellert
    Abstract:

    Mammalian cells are protected from the effects of DNA double-strand breaks by end-joining repair. Cells lacking the XRCC4 Protein are hypersensitive to agents that induce DNA double-strand breaks, and are unable to complete V(D)J recombination. The residual repair of broken DNA ends in XRCC4-deficient cells requires short sequence homologies, thus possibly implicating XRCC4 in end alignment. We show that XRCC4 binds DNA, and prefers DNA with nicks or broken ends. XRCC4 also binds to DNA ligase IV and enhances its joining activity. This stimulatory effect is shown to occur at the adenylation of the enzyme. DNA binding of XRCC4 is correlated with its complementation of the V(D)J recombination defects in XRCC4-deficient cells, but is not required for stimulation of DNA ligase IV. Thus, the ability of XRCC4 to bind to DNA suggests functions independent of DNA ligase IV.

Patrick Calsou - One of the best experts on this subject based on the ideXlab platform.

  • dna dependent Protein kinase and XRCC4 dna ligase iv mobilization in the cell in response to dna double strand breaks
    Journal of Biological Chemistry, 2005
    Co-Authors: Jerome Drouet, Christine Delteil, Jacques Lefrancois, Patrick Concannon, Bernard Salles, Patrick Calsou
    Abstract:

    Abstract Repair of DNA double strand breaks (DSBs) by the non-homologous end joining (NHEJ) pathway in mammals requires at least the DNA-dependent Protein kinase (DNA-PK) and the DNA ligase IV-XRCC4 Protein complexes. DNA-PK comprises the Ku70/Ku80 heterodimer and the catalytic subunit DNA-PKcs. Here we report the first description of the nuclear mobilization of endogenous NHEJ Proteins after exposure of human cells to double strand-breaking agents. DSB infliction specifically induced a dose- and time-dependent mobilization of Ku70/80, DNA-PKcs, XRCC4, and DNA ligase IV Proteins from a soluble nucleoplasmic compartment to a less extractable nuclear fraction. XRCC4 recruitment was accompanied by its DNA-PK-dependent phosphorylation. The recruited Proteins co-immunoprecipitated, indicating that they had assembled into complexes. However, DNA-PK was attached to chromatin, whereas XRCC4-ligase IV resisted solubilization by DNase I. The rates of appearance and dissolution of NHEJ Proteins paralleled that of histone variant H2AX phosphorylation and dephosphorylation. We established that under conditions of genomic DSB infliction 1) Ku recruitment was not dependent on the co-recruitment of the other NHEJ Proteins, 2) DNA-PKcs was physically required for the mobilization of the XRCC4-ligase IV complex, 3) DNA ligase IV was physically necessary for stable recruitment of XRCC4, and 4) phosphorylation of either H2AX or XRCC4 was unnecessary for DNA-PK or XRCC4-ligase IV recruitment. Altogether these results offer insights into the interplay between key NHEJ Proteins during this repair process in the cell.

Mauro Modesti - One of the best experts on this subject based on the ideXlab platform.

  • Tetramerization and DNA ligase IV interaction of the DNA double-strand break repair Protein XRCC4 are mutually exclusive.
    Journal of Molecular Biology, 2003
    Co-Authors: Mauro Modesti, Martin Gellert, Murray S. Junop, Rodolfo Ghirlando, Wei Yang, Mandy W.m.m. Van De Rakt, Roland Kanaar
    Abstract:

    The XRCC4 Protein is of critical importance for the repair of broken chromosomal DNA by non-homologous end joining (NHEJ). The absence of XRCC4 abolishes chromosomal NHEJ almost completely. One reason for this severe phenotype is that XRCC4 binds and modulates the stability and activity of the NHEJ-specific ligase, DNA ligase IV. XRCC4 in solution is in equilibrium between the dimeric and tetrameric forms. Previous structural studies have shown that the interface between dimers is located in the same region as that implicated in DNA ligase IV interaction. With the use of equilibrium sedimentation analysis, we show here that only the XRCC4 dimer can associate with DNA ligase IV, forming a monodisperse complex of 2:1 stoichiometry in solution. In addition, physical analysis of XRCC4/DNA ligase IV complex formation, combined with mutational analysis of XRCC4, indicates that tetramerization and DNA ligase IV binding are mutually exclusive. We propose that the putative function of the XRCC4 tetramer is distinct from its DNA ligase IV-associated function.

  • Crystal structure of the XRCC4 DNA repair Protein and implications for end joining.
    The EMBO Journal, 2000
    Co-Authors: Murray S. Junop, Mauro Modesti, Martin Gellert, Alba Guarné, Rodolfo Ghirlando, Wei Yang
    Abstract:

    XRCC4 is essential for carrying out non‐homologous DNA end joining (NHEJ) in all eukaryotes and, in particular, V(D)J recombination in vertebrates. XRCC4 Protein forms a complex with DNA ligase IV that rejoins two DNA ends in the last step of V(D)J recombination and NHEJ to repair double strand breaks. XRCC4‐defective cells are extremely sensitive to ionizing radiation, and disruption of the XRCC4 gene results in embryonic lethality in mice. Here we report the crystal structure of a functional fragment of XRCC4 at 2.7 A resolution. XRCC4 Protein forms a strikingly elongated dumb‐bell‐like tetramer. Each of the N‐terminal globular head domains consists of a β‐sandwich and a potentially DNA‐binding helix– turn–helix motif. The C‐terminal stalk comprising a single α‐helix >120 A in length is partly incorporated into a four‐helix bundle in the XRCC4 tetramer and partly involved in interacting with ligase IV. The XRCC4 structure suggests a possible mode of coupling ligase IV association with DNA binding for effective ligation of DNA ends.

  • dna binding of XRCC4 Protein is associated with v d j recombination but not with stimulation of dna ligase iv activity
    The EMBO Journal, 1999
    Co-Authors: Mauro Modesti, Joanne E Hesse, Martin Gellert
    Abstract:

    Mammalian cells are protected from the effects of DNA double-strand breaks by end-joining repair. Cells lacking the XRCC4 Protein are hypersensitive to agents that induce DNA double-strand breaks, and are unable to complete V(D)J recombination. The residual repair of broken DNA ends in XRCC4-deficient cells requires short sequence homologies, thus possibly implicating XRCC4 in end alignment. We show that XRCC4 binds DNA, and prefers DNA with nicks or broken ends. XRCC4 also binds to DNA ligase IV and enhances its joining activity. This stimulatory effect is shown to occur at the adenylation of the enzyme. DNA binding of XRCC4 is correlated with its complementation of the V(D)J recombination defects in XRCC4-deficient cells, but is not required for stimulation of DNA ligase IV. Thus, the ability of XRCC4 to bind to DNA suggests functions independent of DNA ligase IV.

Jerome Drouet - One of the best experts on this subject based on the ideXlab platform.

  • dna dependent Protein kinase and XRCC4 dna ligase iv mobilization in the cell in response to dna double strand breaks
    Journal of Biological Chemistry, 2005
    Co-Authors: Jerome Drouet, Christine Delteil, Jacques Lefrancois, Patrick Concannon, Bernard Salles, Patrick Calsou
    Abstract:

    Abstract Repair of DNA double strand breaks (DSBs) by the non-homologous end joining (NHEJ) pathway in mammals requires at least the DNA-dependent Protein kinase (DNA-PK) and the DNA ligase IV-XRCC4 Protein complexes. DNA-PK comprises the Ku70/Ku80 heterodimer and the catalytic subunit DNA-PKcs. Here we report the first description of the nuclear mobilization of endogenous NHEJ Proteins after exposure of human cells to double strand-breaking agents. DSB infliction specifically induced a dose- and time-dependent mobilization of Ku70/80, DNA-PKcs, XRCC4, and DNA ligase IV Proteins from a soluble nucleoplasmic compartment to a less extractable nuclear fraction. XRCC4 recruitment was accompanied by its DNA-PK-dependent phosphorylation. The recruited Proteins co-immunoprecipitated, indicating that they had assembled into complexes. However, DNA-PK was attached to chromatin, whereas XRCC4-ligase IV resisted solubilization by DNase I. The rates of appearance and dissolution of NHEJ Proteins paralleled that of histone variant H2AX phosphorylation and dephosphorylation. We established that under conditions of genomic DSB infliction 1) Ku recruitment was not dependent on the co-recruitment of the other NHEJ Proteins, 2) DNA-PKcs was physically required for the mobilization of the XRCC4-ligase IV complex, 3) DNA ligase IV was physically necessary for stable recruitment of XRCC4, and 4) phosphorylation of either H2AX or XRCC4 was unnecessary for DNA-PK or XRCC4-ligase IV recruitment. Altogether these results offer insights into the interplay between key NHEJ Proteins during this repair process in the cell.