DNA Ligase IV regulates XRCC4 nuclear localization
Introduction
Double strand breaks (DSBs) are one of the most deleterious lesions that can occur within the genome of the cell. These lesions can arise as a result of normal physiological cellular processes, such as V(D)J recombination and class switch recombination during immune cell development [1], [2]. DSBs are also generated during ionizing radiation (IR) and production of oxidative free radicals [3]. In mammalian cells, two major pathways have evolved for the repair of DSBs, namely, homologous recombination (HR) and non-homologous end joining (NHEJ) [4], [5], [6]. HR is a homology dependent reaction and requires the presence of a sister chromatid or homologous chromosome, which functions as a DNA template; this is the main functional pathway during late S/G2 phase of the cell cycle. In contrast, NHEJ, because of its homology-independence, is active throughout the cell cycle but has been found to predominate during G1. Repair by classical NHEJ is considered as error-prone due to the frequent loss or addition of nucleotides at the site of the DSB. However, despite its mutagenic properties the NHEJ pathway is the major pathway utilized to repair DSB, including those that arise as a result of somatic recombination during the development and maturation of immune cells.
Repair via NHEJ involves several core factors including Ku70/80, DNA-PKcs, Artemis, XLF, XRCC4 and DNA Ligase IV (referred to as Ligase IV for the rest of the text). The Ku70/80 heterodimer senses and recognizes breaks in chromosomal DNA and together with DNA-PKcs, stabilize the free ends. Artemis, an endonuclease, along with polymerases λ, μ (PolX family) and terminal deoxynucleotidyl transferase (TdT), play important roles in the processing of DNA ends making them ready for ligation. Finally, the Ligase IV/XRCC4/XLF complex completes ligation and resolves the DSB [7], [8].
Ligase IV, in complex with XRCC4 and XLF, is indispensable to the NHEJ reaction and absence of either of these factors leads to an impaired ability to repair DSBs and immunodeficiency [9], [10], [11]. Hypomorphic mutations within the Ligase IV gene, which disrupt protein function result in partial immunodeficiency and increased sensitivity to IR, reflecting the deregulated function of the NHEJ machinery [12]. Despite significant progress demonstrating how XLF and XRCC4 regulate Ligase IV function, little is known about how Ligase IV regulates NHEJ. It has been shown that proteasome mediated degradation of Ligase IV prevents the binding of XRCC4 and XLF to DNA, without changing their protein levels [13]. DNA binding by XRCC4 and ligation activity of the complex was restored following complementation with the full length Ligase IV [13]. Independent studies showed that localization of XRCC4 and XLF to chromatin was also dependent on Ligase IV [14], [15]. Ligase IV C-terminal region was sufficient to drive localization of XRCC4 to chromatin [16]. Additionally, while XLF is known to interact directly with XRCC4, an intact Ligase IV/XRCC4 complex is needed for the appropriate recruitment of XLF to chromatin and for its efficient interaction with XRCC4 [15]. The Ligase IV/XRCC4 complex contributes to DNA-PKcs autophosphorylation as well as DNA-PKcs mediated DNA end synapsis [17]. A role for the Ligase IV/XRCC4 complex in recruiting and/or modulating the activity of processing enzymes, including nucleases and polymerases, was also suggested [18], [19], [20], [21]. These findings indicate that Ligase IV is critical to the recruitment, assembly and function of the processing and ligation complexes at the site of DSBs. However, the mechanism(s) by which Ligase IV functions to control NHEJ and NHEJ factors remains poorly characterized.
Here, we report that in the absence of Ligase IV, XRCC4 accumulates in the cytoplasm and this retention is independent of DNA damage. Specifically, the C-terminal of Ligase IV plays an important role in regulating the subcellular localization of XRCC4. In addition, human fibroblasts from LIG4 syndrome patients showed a large decrease in XRCC4 protein levels. Together our data suggest new mechanisms by which Ligase IV contributes to the regulation of its protein partner, XRCC4.
Section snippets
Antibodies
Rabbit polyclonal antibodies: anti-Ligase IV against amino acids 1–240, anti-XRCC4 (Serotec), anti-Artemis raised against full-length recombinant Artemis, anti-XLF (Bethyl Laboratories, Inc.), anti-DNA-PKcs (Santa Cruz Biotechnology, Inc.), anti-Ku70, anti-Ku80 (Santa Cruz Biotechnology, Inc.) and anti-Phospho-p53 (Ser315) (Cell Signaling). Goat polyclonal antibodies: anti-Lamin B1 (Santa Cruz Biotechnology, Inc.). Mouse monoclonal antibodies: anti-Flag M2 (Sigma–Aldrich), anti-p53 (BioLegend),
Absence of Ligase IV results in cytoplasmic accumulation of XRCC4, an effect that is not altered upon induction of DNA damage
Due to the reported defect in XRCC4 and XLF recruitment to chromatin in the absence of Ligase IV [14], [15], we hypothesized that loss of Ligase IV would affect the nuclear localization of one or both of these core factors. To investigate our hypothesis, two human pre-B cell lines were utilized, Nalm 6 (WT) and the Ligase IV deficient line, N114P2 (Lig IV −/−) [22]. Nuclear and cytoplasmic fractions were prepared from both WT and Lig IV −/− cells. These fractions were analyzed by immunoblot for
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
Conflict of interest statement
The authors declare no conflict of interest.
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
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Present address: Department of Melanoma Medical Oncology, University of Texas MD Anderson Cancer Center, Houston, TX 77054, United States.