Crystal structure of human XLF: A twist in nonhomologous DNA end-joining

Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, ON L8N 3Z5, Canada.
Molecular Cell (Impact Factor: 14.02). 01/2008; 28(6):1093-101. DOI: 10.1016/j.molcel.2007.10.024
Source: PubMed


DNA double-strand breaks represent one of the most severe forms of DNA damage in mammalian cells. One pathway for repairing these breaks occurs via nonhomologous end-joining (NHEJ) and depends on XRCC4, LigaseIV, and Cernunnos, also called XLF. Although XLF stimulates XRCC4/LigaseIV to ligate mismatched and noncohesive DNA ends, the mechanistic basis for this function remains unclear. Here we report the structure of a partially functional 224 residue N-terminal fragment of human XLF. Despite only weak sequence similarity, XLF(1-170) shares structural homology with XRCC4(1-159). However, unlike the highly extended 130 A helical domain observed in XRCC4, XLF adopts a more compact, folded helical C-terminal region involving two turns and a twist, wrapping back to the structurally conserved N terminus. Mutational analysis of XLF and XRCC4 reveals a potential interaction interface, suggesting a mechanism for how XLF stimulates the ligation of mismatched ends.

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    • "The XLF CTR region is required for recruitment of XLF to Ku at the DSB, stabilizing the interaction with XRCC4 [38]. Right: The initial Ku-XLF-XRCC4 complex may initiate XLF-XRCC4 filament formation [30] [75] [135] [136] and create an extended grooved scaffold to stabilize DSBs [75]. (B) Left: Phosphorylation of XRCC4 promotes a high affinity interaction with the FHA domain of PNKP (orange). "
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    ABSTRACT: Non-homologous end joining (NHEJ) is the major pathway for repair of DNA double-strand breaks (DSBs) in human cells. NHEJ is also needed for V(D)J recombination and the development of T and B cells in vertebrate immune systems, and acts in both the generation and prevention of non-homologous chromosomal translocations, a hallmark of genomic instability and many human cancers. X-ray crystal structures, cryo-electron microscopy envelopes, and small angle X-ray scattering (SAXS) solution conformations and assemblies are defining most of the core protein components for NHEJ: Ku70/Ku80 heterodimer; the DNA dependent protein kinase catalytic subunit (DNA-PKcs); the structure-specific endonuclease Artemis along with polynucleotide kinase/phosphatase (PNKP), aprataxin and PNKP related protein (APLF); the scaffolding proteins XRCC4 and XLF (XRCC4-like factor); DNA polymerases, and DNA ligase IV (Lig IV). The dynamic assembly of multi-protein NHEJ complexes at DSBs is regulated in part by protein phosphorylation. The basic steps of NHEJ have been biochemically defined to require: (1) DSB detection by the Ku heterodimer with subsequent DNA-PKcs tethering to form the DNA-PKcs-Ku-DNA complex (termed DNA-PK), (2) lesion processing, and (3) DNA end ligation by Lig IV, which functions in complex with XRCC4 and XLF. The current integration of structures by combined methods is resolving puzzles regarding the mechanisms, coordination and regulation of these three basic steps. Overall, structural results suggest the NHEJ system forms a flexing scaffold with the DNA-PKcs HEAT repeats acting as compressible macromolecular springs suitable to store and release conformational energy to apply forces to regulate NHEJ complexes and the DNA substrate for DNA end protection, processing, and ligation.
    DNA repair 05/2014; 17. DOI:10.1016/j.dnarep.2014.02.009 · 3.11 Impact Factor
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    • "Structural studies of XLF and the XRCC4/XLF complex provide powerful starting points for an investigation of the functional mechanism of XLF. Despite the low sequence identity, the crystal structures of XLF and XRCC4 demonstrate that the two proteins are homologous homodimers comprising globular head domains and C-terminal helices that form coiled-coil tail structures [29] [30] [31] [32]. "
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    ABSTRACT: Non-homologous end joining (NHEJ) repairs DNA double-strand breaks generated by DNA damage and also those occurring in V(D)J recombination in immunoglobulin and T cell receptor production in the immune system. In NHEJ DNA-PKcs assembles with Ku heterodimer on the DNA ends at double-strand breaks, in order to bring the broken ends together and to assemble other proteins, including DNA ligase IV (LigIV), required for DNA repair. Here we focus on structural aspects of the interactions of LigIV with XRCC4, XLF, Artemis and DNA involved in the bridging and end-joining steps of NHEJ. We begin with a discussion of the role of XLF, which interacts with Ku and forms a hetero-filament with XRCC4; this likely forms a scaffold bridging the DNA ends. We then review the well-defined interaction of XRCC4 with LigIV, and discuss the possibility of this complex interrupting the filament formation, so positioning the ligase at the correct positions close to the broken ends. We also describe the interactions of LigIV with Artemis, the nuclease that prepares the ends for ligation and also interacts with DNA-PK. Lastly we review the likely affects of Mendelian mutations on these multiprotein assemblies and their impacts on the form of inherited disease.
    DNA repair 05/2014; 17(100). DOI:10.1016/j.dnarep.2014.02.010 · 3.11 Impact Factor
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    • "The finding that phosphorylation of the CTRs of XRCC4 and XLF is functionally redundant may help to explain some of the discrepancies in the literature on the importance of the XRCC4 and XLF CTRs in NHEJ. Although the XRCC4 CTR and the XLF CTR are both required for DNA binding in vitro (Andres et al. 2007; Yu et al. 2008) and the XLF CTR is required for recruitment of XLF to sites of UV-laser-induced damage in vivo (Yano et al. 2011), both CTRs are dispensable for cell survival following IR as well as V(D)J recombination in vivo (Leber et al. 1998; Malivert et al. 2009; Modesti et al. 1999). However, it may be that the CTR of either XRCC4 or XLF can compensate for loss of the other. "
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    ABSTRACT: DNA double strand breaks (DSBs), induced by ionizing radiation (IR) and endogenous stress including replication failure, are the most cytotoxic form of DNA damage. In human cells, most IR-induced DSBs are repaired by the nonhomologous end joining (NHEJ) pathway. One of the most critical steps in NHEJ is ligation of DNA ends by DNA ligase IV (LIG4), which interacts with, and is stabilized by, the scaffolding protein X-ray cross-complementing gene 4 (XRCC4). XRCC4 also interacts with XRCC4-like factor (XLF, also called Cernunnos); yet, XLF has been one of the least mechanistically understood proteins and precisely how XLF functions in NHEJ has been enigmatic. Here, we examine current combined structural and mutational findings that uncover integrated functions of XRCC4 and XLF and reveal their interactions to form long, helical protein filaments suitable to protect and align DSB ends. XLF-XRCC4 provides a global structural scaffold for ligating DSBs without requiring long DNA ends, thus ensuring accurate and efficient ligation and repair. The assembly of these XRCC4-XLF filaments, providing both DNA end protection and alignment, may commit cells to NHEJ with general biological implications for NHEJ and DSB repair processes and their links to cancer predispositions and interventions.
    Biochemistry and Cell Biology 02/2013; 91(1):31-41. DOI:10.1139/bcb-2012-0058 · 2.15 Impact Factor
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