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  • alkylation of dna br Results br Discussion Repair by NHEJ im


    Discussion Repair by NHEJ implicitly requires the pairing together of broken chromosome ends. A complex of Ku, XRCC4, DNA ligase IV, and XLF (PEC) is necessary and sufficient for this purpose (Reid et al., 2015). Here, we describe dynamic changes in this complex that are triggered by differences in end structure and show that this response is essential for efficient cellular repair.
    Experimental Procedures
    Author Contributions
    Acknowledgments We thank Kaitlyn Walsh for performing experiments related to this work. We thank Dr. Eric Hendrickson for providing LIG4+/+ and LIG4−/− cell lines used in this work. M.P.C. was supported by the National Cancer Institute (F31CA203156) and the National Institute of General Medical Sciences (T32GM007092). M.P.C., D.A. Ramsden, and G.W.S. were supported by the National Cancer Institute (CA084442). E.R. and D.A. Reid were supported by the National Institute of General Medical Sciences (GM108118). M.R.L., H.H.C., and G.W.S. were supported by the National Cancer Institute (CA100504) and the National Institute of General Medical Sciences (GM118009).
    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 alkylation of dna 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.