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  • br Material and methods br Results br Discussion Ligase


    Material and methods
    Discussion Ligase IV is essential for the final ligation of DSBs through the NHEJ pathway. Its co-factor, XRCC4, has been shown to be important for increasing its activity and stability. Interaction between these two factors have been mapped to the linker region between the two BRCT domains found in the Ligase IV C-terminal known as the XIR and a portion of the BRCT II, which is necessary to stabilize the interaction [41], [43]. In this work, we highlight a novel role for Ligase IV as an important regulator of the NHEJ reaction in controlling the nuclear localization of its co-factors XRCC4 and XLF. Ligase IV might facilitate efficient nuclear localization of XRCC4 by increasing its nuclear retention and/or by allowing its nuclear translocation. While future work will be required to establish the precise mechanism, our findings provide an explanation for the impaired recruitment of XRCC4 and XLF to NK 252 that has been previously observed [14], [15]. We also highlight the role of Ligase IV BRCT I domain, along with the XIR, in the nuclear localization of XRCC4. Our findings suggest that the Ligase IV/XRCC4 complex is formed in the cytoplasm and subsequently controls the organization of a repair complex within the nucleus. Structural and biochemical studies have revealed that together with XRCC4, XLF forms long filamentous structures that are important for the bridging of the two broken ends during ligation [47], [48], [49]. Furthermore, DNA-PKcs serves to regulate this complex [50]. Published work has shown that Ligase IV is essential for DNA-PKcs autophosphorylation and DNA end synapsis [17]; these studies, together with our findings, suggest that Ligase IV has a critical, yet largely uncharacterized role in the assembly of the nuclear DNA repair complexes. Two mechanisms have been suggested to control the nuclear localization of XRCC4, a nuclear localization signal present in XRCC4 (between aa 270 and 275) and the SUMO modification of the protein at lysine 210 [51], [52]. In this study, we show that Ligase IV allows nuclear localization of XRCC4 and in doing so, indirectly facilitates nuclear accumulation of XLF. Along with other NHEJ factors, such as DNA PKcs, this complex orchestrated by Ligase IV would promote repair via non-homologous end joining. Interestingly, in our cellular fractionation experiments, we observed a consistent difference in the migration pattern of the nuclear and cytoplasmic XRCC4 protein. Although earlier studies have demonstrated the phosphorylation and monoubiquitination of XRCC4, the role of these posttranslational modifications remains largely uncharacterized [21], [53], [54], [55], [56]. We postulate that these modifications might also contribute to regulate the nuclear cytoplasmic distribution of XRCC4. How the various mechanisms that control nuclear localization and/or retention of XRCC4 in the NK 252 nucleus work together to ensure efficient and regulated NHEJ will be the subject of future studies. Human mutations in the Ligase IV gene have been identified in patients with LIG4 syndrome, as well as in patients with Dubowitz syndrome [12], [13]. These patients present a wide range of defects, including, high levels of chromosomal breaks, predisposition to tumor development and variable immune defects. The analysis of Ligase IV hypomorphs, presented in this manuscript, sheds light on the sensitivity to IR and defects in V(D)J recombination observed in patients with hypomorphic mutations within the Ligase IV gene. Our work shows that Ligase IV is not only critical for proper nuclear localization of XRCC4 but also appears to have a role in stabilizing XRCC4 in primary human fibroblasts. The difference in XRCC4 stability in human preB cells versus in human primary fibroblasts is still unclear but raises intriguing questions that deserve further investigation.
    Conflict of interest statement
    Acknowledgements We thank members of the Cortes laboratory for helpful discussions and reagents and Dr. Juan Carcamo for constructive suggestions and critical reading of the manuscript. We thank Dr. Michael R. Lieber for providing the Nalm 6 and N114P2 cells, Dr. Anna Villa for the control fibroblast NM-1. Dr. Tomas Lindahl and Dr. Michael Lieber provided the cDNA for human Ligase IV. The advice and equipment provided by the Mount Sinai Microscopy Shared Resource Facility were instrumental to perform the described studies. Work in the P.C. laboratory is supported by the National Institutes of Health (R01 AI080755 and R01 AI070880 from NIH). D.B.F. was supported by pre-doctoral training grants given to the Immunology Institute by the National Institute of Health (T32-A1007605-09 and 5T32A1007605-10).