Academic literature on the topic 'Ligase IV/XRCC4'

ABSTRACT Genetic experiments have determined that Ku, XRCC4, and ligase IV are required for repair of double-strand breaks by the end-joining pathway. The last two factors form a tight complex in cells. However, ligase IV is only one of three known mammalian ligases and is intrinsically the least active in intermolecular ligation; thus, the biochemical basis for requiring this ligase has been unclear. We demonstrate here a direct physical interaction between the XRCC4-ligase IV complex and Ku. This interaction is stimulated once Ku binds to DNA ends. Since XRCC4-ligase IV alone has very low DNA binding activity, Ku is required for effective recruitment of this ligase to DNA ends. We further show that this recruitment is critical for efficient end-joining activity in vitro. Preformation of a complex containing Ku and XRCC4-ligase IV increases the initial ligation rate 20-fold, indicating that recruitment of the ligase is an important limiting step in intermolecular ligation. Recruitment by Ku also allows XRCC4-ligase IV to use Ku's high affinity for DNA ends to rapidly locate and ligate ends in an excess of unbroken DNA, a necessity for end joining in cells. These properties are conferred only on ligase IV, because Ku does not similarly interact with the other mammalian ligases. We have therefore defined cell-free conditions that reflect the genetic requirement for ligase IV in cellular end joining and consequently can explain in molecular terms why this factor is required.

ABSTRACT Nonhomologous end-joining represents the major pathway used by human cells to repair DNA double-strand breaks. It relies on the XRCC4/DNA ligase IV complex to reseal DNA strands. Here we report the high-resolution crystal structure of human XRCC4 bound to the carboxy-terminal tandem BRCT repeat of DNA ligase IV. The structure differs from the homologous Saccharomyces cerevisiae complex and reveals an extensive DNA ligase IV binding interface formed by a helix-loop-helix structure within the inter-BRCT linker region, as well as significant interactions involving the second BRCT domain, which induces a kink in the tail region of XRCC4. We further demonstrate that interaction with the second BRCT domain of DNA ligase IV is necessary for stable binding to XRCC4 in cells, as well as to achieve efficient dominant-negative effects resulting in radiosensitization after ectopic overexpression of DNA ligase IV fragments in human fibroblasts. Together our findings provide unanticipated insight for understanding the physical and functional architecture of the nonhomologous end-joining ligation complex.

DNA Ligase IV is responsible for the repair of DNA double-strand breaks (DSB), including DSBs that are generated during V(D)J recombination. Like other DNA ligases, Ligase IV contains a catalytic core with three subdomains—the DNA binding (DBD), the nucleotidyltransferase (NTD), and the oligonucleotide/oligosaccharide-fold subdomain (OBD). Ligase IV also has a unique C-terminal region that includes two BRCT domains, a nuclear localization signal sequence and a stretch of amino acid that participate in its interaction with XRCC4. Out of the three mammalian ligases, Ligase IV is the only ligase that participates in and is required for V(D)J recombination. Identification of the minimal domains within DNA Ligase IV that contribute to V(D)J recombination has remained unresolved. The interaction of the Ligase IV DNA binding domain with Artemis, and the interaction of its C-terminal region with XRCC4, suggest that both of these regions that also interact with the Ku70/80 heterodimer are important and might be sufficient for mediating participation of DNA Ligase IV in V(D)J recombination. This hypothesis was investigated by generating chimeric ligase proteins by swapping domains, and testing their ability to rescue V(D)J recombination in Ligase IV-deficient cells. We demonstrate that a fusion protein containing Ligase I NTD and OBDs flanked by DNA Ligase IV DBD and C-terminal region is sufficient to support V(D)J recombination. This chimeric protein, which we named Ligase 37, complemented formation of coding and signal joints. Coding joints generated with Ligase 37 were shorter than those observed with wild type DNA Ligase IV. The shorter length was due to increased nucleotide deletions and decreased nucleotide insertions. Additionally, overexpression of Ligase 37 in a mouse pro-B cell line supported a shift towards shorter coding joints. Our findings demonstrate that the ability of DNA Ligase IV to participate in V(D)J recombination is in large part mediated by its DBD and C-terminal region.

ABSTRACT Mammalian DNA polymerase μ (pol μ) is related to terminal deoxynucleotidyl transferase, but its biological role is not yet clear. We show here that after exposure of cells to ionizing radiation (IR), levels of pol μ protein increase. pol μ also forms discrete nuclear foci after IR, and these foci are largely coincident with IR-induced foci of γH2AX, a previously characterized marker of sites of DNA double-strand breaks. pol μ is thus part of the cellular response to DNA double-strand breaks. pol μ also associates in cell extracts with the nonhomologous end-joining repair factor Ku and requires both Ku and another end-joining factor, XRCC4-ligase IV, to form a stable complex on DNA in vitro. pol μ in turn facilitates both stable recruitment of XRCC4-ligase IV to Ku-bound DNA and ligase IV-dependent end joining. In contrast, the related mammalian DNA polymerase β does not form a complex with Ku and XRCC4-ligase IV and is less effective than pol μ in facilitating joining mediated by these factors. Our data thus support an important role for pol μ in the end-joining pathway for repair of double-strand breaks.

Several findings have revealed a likely role for DNA ligase IV, and interacting protein XRCC4, in the final steps of mammalian DNA double-strand break repair. Recent evidence suggests that the human DNA ligase IV protein plays a critical role in the maintenance of genomic stability. To identify protein–protein interactions that may shed further light on the molecular mechanisms of DSB repair and the biological roles of human DNA ligase IV, we have used the yeast two-hybrid system in conjunction with traditional biochemical methods. These efforts have resulted in the identification of a physical association between the DNA ligase IV polypeptide and the human condensin subunit known as hCAP-E. The hCAP-E polypeptide, a member of the Structural Maintenance of Chromosomes (SMC) super-family of proteins, coimmunoprecipitates from cell extracts with DNA ligase IV. Immunofluorescence studies reveal colocalization of DNA ligase IV and hCAP-E in the interphase nucleus, whereas mitotic cells display colocalization of both polypeptides on mitotic chromosomes. Strikingly, the XRCC4 protein is excluded from the area of mitotic chromosomes, suggesting the formation of specialized DNA ligase IV complexes subject to cell cycle regulation. We discuss our findings in light of known and hypothesized roles for ligase IV and the condensin complex.

The classic nonhomologous end-joining (c-NHEJ) pathway is largely responsible for repairing double-strand breaks (DSBs) in mammalian cells. XLF stimulates the XRCC4/DNA ligase IV complex by an unknown mechanism. XLF interacts with XRCC4 to form filaments of alternating XRCC4 and XLF dimers that bridge DNA endsin vitro, providing a mechanism by which XLF might stimulate ligation. Here, we characterize two XLF mutants that do not interact with XRCC4 and cannot form filaments or bridge DNAin vitro. One mutant is fully sufficient in stimulating ligation by XRCC4/Lig4in vitro; the other is not. This separation-of-function mutant (which must function as an XLF homodimer) fully complements the c-NHEJ deficits of some XLF-deficient cell strains but not others, suggesting a variable requirement for XRCC4/XLF interaction in living cells. To determine whether the lack of XRCC4/XLF interaction (and potential bridging) can be compensated for by other factors, candidate repair factors were disrupted in XLF- or XRCC4-deficient cells. The loss of either ATM or the newly described XRCC4/XLF-like factor, PAXX, accentuates the requirement for XLF. However, in the case of ATM/XLF loss (but not PAXX/XLF loss), this reflects a greater requirement for XRCC4/XLF interaction.

Abstract Aberrant class switch recombination (CSR), the physiological process that regulates maturation of the antibody response, is believed to be an early event in the pathogenesis of myeloma. The genetic basis of CSR, from initiation of the DNA double-strand break through to detection and repair, has been elucidated. We hypothesised that germline polymorphisms in the genes implicated in DNA double strand break repair may contribute to susceptibility to myeloma. We therefore assessed 32 SNPs in 3 genes central to the DNA repair pathway in patients with myeloma and controls from the EpiLymph Study, a European study of the epidemiology of lymphoid neoplasms, and from an Irish hospital registry (306 cases, 263 controls). The genes examined were XRCC3, XRCC4, and XRCC5. XRCC3 is a member of the RecA/Rad51-related protein family that participates in homologous recombination. The XRCC4 protein forms a complex with DNA ligase IV and DNA-dependent protein kinase in the repair of DNA double-strand breaks by non-homologous end joining. XRCC5 encodes the 80-kilodalton subunit of the Ku heterodimer protein, the DNA-binding component of the DNA-dependent protein kinase. SNPs from the chosen genes were identified from HapMap CEU Phase I and II genotype data (Public Release #20; 2006-01-24). Haplotype-tagging SNPs (htSNPs) were chosen based on Tagger analysis (as implemented in Haploview Version 3.2). A SNP in GSTP1 was also genotyped to allow for comparison with allele frequencies previously generated from a myeloma cohort. Genotyping was performed using TaqMan®-based assays on the 7900 ABI HT platform. The drop-out rate was consistently less than 3% in all assays. For quality control (QC) purposes, duplicates of 10% of the samples were interspersed throughout the plates. The concordance rates for QC samples were greater than 98%. GSTP1 SNP results were comparable to previously published findings. For the htSNP rs963248 in XRCC4, Allele A was significantly more frequent in cases than in controls (86.4% vs.80.8%) (p=0.0105), as was the AA genotype (74% vs. 65%) (p=0.026). Haplotype analysis was performed using Cocaphase for rs963248 in combination with additional SNPs in XRCC4. The strongest evidence of association came from the A - T haplotype from rs963248-rs2891980 (80.9% vs. 74.5%; p=0.008). For XRCC5, the genotype GG from rs1051685 was detected in 10 cases from different national populations but in only 1 control (p=0.015). Interestingly, this SNP is located in the 3′ UTR of XRCC5. Overall, these data provide support for the hypothesis that common variation in the genes encoding DNA repair proteins contributes to susceptibility to myeloma.