Activity of DNA ligase IV stimulated by complex formation with XRCC4 protein in mammalian cells

Abstract

Mutation of the XRCC4 gene in mammalian cells prevents the formation of the signal and coding joints in the V(D)J recombination reaction, which is necessary for production of a functional immunoglobulin gene, and renders the cells highly sensitive to ionizing radiation. However, XRCC4 shares no sequence homology with other proteins, nor does it have a biochemical activity to indicate what its function might be. Here we show that DNA ligase IV 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.

Severe combined immunodeficiency and microcephaly in siblings with hypomorphic mutations in DNA ligase IV

“…Protein complexes were resolved by 10% SDS-PAGE and the gel was stained with Coomassie Brilliant Blue R250.XRCC4 migrates as two bands [36]. (B) DNA ligation assay with the WT and the Q280R DNA ligase IV complex.…”

“…Forty-eight hours after the transfection, cells were infected with vaccinia virus vTF7-3, which expresses the T7 polymerase, and v12-2-2 that expresses the WT 9xHis and hemagglutinin-tagged XRCC4.Twenty-four hours after the infection, cells were harvested, and the WT and mutant DNA ligase IV/XRCC4 complexes were immunoprecipitated as described [8]. The ligation substrate was prepared by annealing the end-labeled 18 nt 5 0 [c-32 P]UG-305 (5 0 -ATCTA-GACGAGGGGAGGG-3 0 ) and an 18 nt UG-304 (5 0 -CGGAC-GAATCCACCCGGG-3 0 ) to the 3 0 and 5 0 portions of an 80-nt UG-301 (5 0 -GGGTGGATTCGTCCGCTTTC CTTCCCTTTTCT-CTCTTTTCCCTTTCTCCTTCCTTCCTTCCCTCCCCTCGTCTA-GATCCC-3 0 ), respectively [36]. Intramolecular ligation occurs when UG-301 bends to allow the two ends of UG-301/304/305 to align with each other (see diagram in Fig.…”

Modes of interaction among yeast Nej1, Lif1 and Dnl4 proteins and comparison to human XLF, XRCC4 and Lig4

“…XRCC4/Lif1 is known from crystallographic studies to be homodimeric, with an Nterminal globular head domain followed by a long ∼100 amino acid coiled-coil of α-helices that provides extensive and stable interaction between the two monomers [10,11]. The Lig4/ Dnl4 BRCT domains bind strongly to XRCC4/Lif1 by encircling this coiled coil at a conserved motif near its C-terminal end [7], supporting robust co-purification of Lig4/Dnl4 with XRCC4/ Lif1 [12][13][14]. Finally, XRCC4/Lif1 contains a C-terminal region that is poorly conserved and structurally uncharacterized, but that is nonetheless phosphorylated and required for NHEJ and interaction with FHA domains of two proteins [15,16] (manuscript in preparation).…”

DNA double-strand break repair and development

“…The extent to which Artemis is required during canonical NHEJ (independent of V(D)J recombination) remains uncertain, as conflicting data on its requirement for end joining have been reported (Rooney et al, 2003;Riballo et al, 2004;Zhang et al, 2004;Wang et al, 2005b). However, Lig4 and Xrcc4 are essential for NHEJ and embryonic survival (Barnes et al, 1998;Frank et al, 1998;Gao et al, 1998b), and their interaction is required for efficient end joining as Lig4 is not sufficient to complete repair in the absence of Xrcc4 (Critchlow et al, 1997;Grawunder et al, 1997Grawunder et al, , 1998aBassing and Alt, 2004). While HR is available for repair during late S/G 2 , NHEJ is most prominently activated in G 1 , G 0 and early S phase of the cell cycle (Rothkamm et al, 2003;Thacker and Zdzienicka, 2004).…”