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  • br Acknowledgments Studies in the


    Acknowledgments Studies in the Tomkinson laboratory on DNA ligase III are supported by research grants from the National Institutes of Health (P01 CA92584 and ES12512 to AET), a grant from the V Foundation and the University of New Mexico Cancer Center.
    Introduction Ionizing radiation (IR) as well as a subset of chemotherapeutic drugs, which are commonly used for cancer therapy, generate various types of DNA damages, among which DNA double-strand break (DSB) is considered most critical. Eukaryotic cells have evolved two major pathways to repair DSBs: homologous recombination and non-homologous end-joining (NHEJ) [1], [2]. In NHEJ, a heterodimer of Ku70 and Ku86 first binds to DSB and in turn recruits DNA-PKcs, which possesses protein kinase activity. When necessary, the DNA end is processed by enzymes, such as Artemis, polynucleotide kinase/phosphatase (PNKP), DNA polymerase λ and μ. Finally, two DNA ends are ligated by DNA Ligase IV (LIG4) in association with XRCC4. XLF (also known as Cernunnos) is thought to stimulate the LIG4 activity toward incompatible or mismatched DNA ends. XRCC4 was initially found as the human cDNA, which could complement the defective V(D)J recombination and radiosensitivity of XR-1 cells, derived from Chinese hamster ovary cell [3]. Subsequently, XRCC4 was found to be associated with LIG4 [4], [5]. XRCC4 stimulates the ligation and adenylation activity of LIG4 [4], [6] and is also required for the stabilization of LIG4 [7]. While XRCC4 consists of 336 amino acids, structural studies indicated that N-terminal part, spanning ∼200 amino acids, forms globular domain and coiled-coil domain, the latter of which mediates dimerization of XRCC4 and its interaction with LIG4 [6], [8], [9]. Although the structure of the remaining C-terminal part of XRCC4, spanning ∼130 amino acids, has not been determined at high resolution, it is deduced from electron microscopy to form a globular structure at the opposite of N-terminal globular head domain [10]. XRCC4 might also have a scaffold role, as it is shown to interact with other repair enzymes like PNKP [11], aprataxin [12] and APLF (aprataxin- and PNK-like factor, also known as PALF, C2orf13 or XIP1) [13], [14]. LIG4 is a 911 amino trans-isomer protein and its N-terminal part, spanning ∼600 amino acids, contains DNA-binding, adenylation and oligo-binding domains, which are thought important for ligase catalytic function. The remaining C-terminal part contains two BRCT (breast cancer associated 1 C-terminal) domains and XRCC4 interacting region (XIR) in between [15]. Recent study showed that XRCC4 and XLF, each as a dimer, interact with their globular head domains to form long, helical filaments, which might bridge or align DNA to facilitate ligation [16]. The mechanisms how these proteins are recruited to DSBs have been explored through various approaches. Nick McElhinny et al. showed Ku-dependent DNA binding of XRCC4/LIG4 by electrophoretic mobility shift assay [17]. Hsu et al. demonstrated binding between Ku and LIG4 and that between XRCC4 and DNA-PKcs by Far-Western analysis [18]. Constantini et al. identified the first BRCT domain of LIG4 as a Ku-binding site [19]. Calsou et al. studied the assembly of proteins on DNA immobilized on paramagnetic beads and showed that Ku and DNA-PKcs were necessary for the recruitment of XRCC4/LIG4 onto DNA [20]. In their later study, they showed DSB-induced insolubilization in cellulo of XRCC4/LIG4, which required DNA-PKcs as well as Ku [21]. In these studies, no effects of wortmannin, a potent inhibitor of DNA-PK, were observed, suggesting that the role of DNA-PKcs might be independent of kinase activity [20], [21]. We reported that radiation-induced chromatin binding of XRCC4 was not abolished by the treatment with wortmannin or the stable expression of DNA-PKcs siRNA, although some reduction was observed [22]. Another emerging approach is the live cell imaging, tracking the behavior of the fluorescently labeled proteins after laser micro irradiation [2]. Mari et al. demonstrated that the accumulation of XRCC4 in irradiated area was dependent on Ku but not on DNA-PKcs [23]. Yano et al. showed that the accumulation of XRCC4 in irradiated area could be observed, but was significantly reduced, in cells lacking DNA-PKcs, indicating the role of DNA-PKcs in stabilizing XRCC4 on chromatin [24]. Moreover, XRCC4 kinetics in kinase-dead DNA-PKcs-expressing cells were similar to normal DNA-PKcs expressing cells, suggesting that DNA-PKcs might play a scaffolding, rather than a catalytic, role [24]. Recent studies by Rulten et al. and Grundy et al. indicated that APLF is recruited to damage site via interaction with Ku and/or PARP-3 and, in turn, promotes the recruitment or retention of XRCC4 [25], [26].