Crystal Structure of Human XLF: A Twist in Nonhomologous DNA End-Joining

Andres, Sara N.; Modesti, Mauro; Tsai, Chun J.; Chu, Gilbert; Junop, M.S.

doi:10.1016/j.molcel.2007.10.024

Cited by 128 publications

(248 citation statements)

References 30 publications

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“…S2D for detailed view of c2 contact region). These results are consistent with an analysis of the domains required for the X4-Cernunnos interaction (16) and with previously reported site-directed mutagenesis studies (27,29), all of which identified the i1 interface, involving the X4 and Cernunnos N-terminal head domains, as the primary interface.…”

Section: Resultssupporting

confidence: 92%

“…This structure is consistent with the mutagenesis data presented by Andres et al, which identified roles for the X4 residues K65 X and K99 X and for the Cernunnos residue L115 C (27). In our Rosetta model, Cernunnos position L115 C is buried in the i1 interface and is in contact with four X4 hydrophobic residues (M59 X , M61 X , F106 X , and L108 X ).…”

Section: Discussionsupporting

confidence: 92%

“…They presented a large cell parameter of 856 Å with a hexagonal symmetry, P6 5 22. Molecular replacement allowed positioning two X4 homodimers (named X4-A and -B) and two Cernunnos homodimers (named Cernunnos-A and -B) in the asymmetric unit, using the individual crystal structures of X4 and Cernunnos (21,27) (see Table S1 for the data collection and refinement statistics).…”

Section: Resultsmentioning

confidence: 99%

“…Cernunnos stimulates the ligase activity of the L4-X4 complex (20,21,(23)(24)(25) and seems particularly important for the ligation of mismatched or noncohesive DNA ends (25,26). Like X4, Cernunnos is a homodimer (27,28). It presents important structural similarities with X4, which include its Author contributions: V.R., P.D., P.L., S.B., J.A., G.F., J.A.M., O.P., R.G., I.C., E.L.C., P.R., J.-P.d.V., and J.-B.C.…”

mentioning

confidence: 99%

“…Both X4 and Cernunnos have C-terminal regions that are predicted to be unstructured. Two-hybrid mapping and mutagenesis studies showed that X4 and Cernunnos interact through their N-terminal head domains (16,27,29). In particular, single mutations at any of three Cernunnos residues (R64, L65, and L115) disrupt its interaction with X4 and abrogate Cernunnos function in several DNA repair assays, despite correct Cernunnos expression and localization (29).…”

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confidence: 99%

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Structural characterization of filaments formed by human Xrcc4–Cernunnos/XLF complex involved in nonhomologous DNA end-joining

Ropars

Drevet

Legrand

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

122

128

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Cernunnos/XLF is a core protein of the nonhomologous DNA end-joining (NHEJ) pathway that processes the majority of DNA double-strand breaks in mammals. Cernunnos stimulates the final ligation step catalyzed by the complex between DNA ligase IV and Xrcc4 (X4). Here we present the crystal structure of the X4 1-157 -Cernunnos 1-224 complex at 5.5-Å resolution and identify the relative positions of the two factors and their binding sites. The X-ray structure reveals a filament arrangement for X4 1-157 and Cernunnos 1-224 homodimers mediated by repeated interactions through their N-terminal head domains. A filament arrangement of the X4-Cernunnos complex was confirmed by transmission electron microscopy analyses both with truncated and full-length proteins. We further modeled the interface and used structure-based site-directed mutagenesis and calorimetry to characterize the roles of various residues at the X4-Cernunnos interface. We identified four X4 residues (Glu 55 , Asp 58 , Met 61 , and Phe 106 ) essential for the interaction with Cernunnos. These findings provide new insights into the molecular bases for stimulatory and bridging roles of Cernunnos in the final DNA ligation step.D NA double-strand breaks (DSBs) are the most toxic DNA lesions in the genome, and unrepaired DSBs can cause large-scale losses of genetic information through chromosome rearrangement (1, 2). These DNA damages result from exposure to exogenous damaging agents, such as ionizing radiation, radiomimetic compounds, and topoisomerase inhibitors. DSBs are also obligate intermediates in several recombination processes in vertebrates, including antigen receptor gene rearrangement, V(D)J recombination (3, 4). In higher eukaryotes, DSBs are repaired by several mechanisms, among which nonhomologous end-joining (NHEJ) represents the major pathway, particularly when sister chromatids are not available (5). Deficiency in the NHEJ machinery results in sensitivity to ionizing radiation and severe combined immune deficiencies in humans and mice due to abortive V(D)J recombination (6). NHEJ is orchestrated by at least seven proteins. The Ku70/Ku80 heterodimer adopts a preformed ring-shaped structure that recognizes and encircles the duplex DNA ends at the DSB (7). Ku70/Ku80 recruits a 469-kDa serine/threonine protein kinase, the DNA-PK catalytic subunit (DNA-PKcs), via a direct interaction and shifts about 10 bp inward so that DNA-PKcs acquires a position at the terminus through its large open-ring cradle structure (8). Upon association with Ku and DNA, DNA-PKcs is activated and phosphorylates several proteins including itself and Ku70/Ku80. The DNA-PK holoenzyme, constituted by Ku and DNA-PKcs, plays a central role in NHEJ. Among other functions, DNA-PK mediates the end-bridging of the DSB extremities (9), regulates access to the DNA ends by processing enzymes such as the DNA-PKcs-associated Artemis nuclease (10, 11), and recruits the Xrcc4-ligase IV complex to DNA ends for the ligation step (12). The Xrcc4-ligase IV complex carries out the final joinin...

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Section: Resultssupporting

confidence: 92%

Section: Discussionsupporting

confidence: 92%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

Structural characterization of filaments formed by human Xrcc4–Cernunnos/XLF complex involved in nonhomologous DNA end-joining

Ropars

Drevet

Legrand

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

122

128

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Non‐homologous end joining: Common interaction sites and exchange of multiple factors in the DNA repair process

Rulten

Grundy

2017

BioEssays

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Non-homologous end-joining (NHEJ) is the dominant means of repairing chromosomal DNA double strand breaks (DSBs), and is essential in human cells. Fifteen or more proteins can be involved in the detection, signalling, synapsis, end-processing and ligation events required to repair a DSB, and must be assembled in the confined space around the DNA ends. We review here a number of interaction points between the core NHEJ components (Ku70, Ku80, DNA-PKcs, XRCC4 and Ligase IV) and accessory factors such as kinases, phosphatases, polymerases and structural proteins. Conserved protein-protein interaction sites such as Ku-binding motifs (KBMs), XLF-like motifs (XLMs), FHA and BRCT domains illustrate that different proteins compete for the same binding sites on the core machinery, and must be spatially and temporally regulated. We discuss how post-translational modifications such as phosphorylation, ADP-ribosylation and ubiquitinylation may regulate sequential steps in the NHEJ pathway or control repair at different types of DNA breaks.

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Clinical variability and novel mutations in the NHEJ1 gene in patients with a Nijmegen breakage syndrome-like phenotype

et al. 2010

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We have previously shown that mutations in the genes encoding DNA Ligase IV (LIGIV) and RAD50, involved in DNA repair by nonhomologous-end joining (NHEJ) and homologous recombination, respectively, lead to clinical and cellular features similar to those of Nijmegen Breakage Syndrome (NBS). Very recently, a new member of the NHEJ repair pathway, NHEJ1, was discovered, and mutations in patients with features resembling NBS were described. Here we report on five patients from four families of different ethnic origin with the NBS-like phenotype. Sequence analysis of the NHEJ1 gene in a patient of Spanish and in a patient of Turkish origin identified homozygous, previously reported mutations, c.168C>G (p.Arg57Gly) and c.532C>T (p.Arg178Ter), respectively. Two novel, paternally inherited truncating mutations, c.495dupA (p.Asp166ArgfsTer20) and c.526C>T (p.Arg176Ter) and two novel, maternal genomic deletions of 1.9 and 6.9 kb of the NHEJ1 gene, were found in a compound heterozygous state in two siblings of German origin and in one Malaysian patient, respectively. Our findings confirm that patients with NBS-like phenotypes may have mutations in the NHEJ1 gene including multiexon deletions, and show that considerable clinical variability could be observed even within the same family.

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Crystal Structure of Human XLF: A Twist in Nonhomologous DNA End-Joining

Cited by 128 publications

References 30 publications

Structural characterization of filaments formed by human Xrcc4–Cernunnos/XLF complex involved in nonhomologous DNA end-joining

Structural characterization of filaments formed by human Xrcc4–Cernunnos/XLF complex involved in nonhomologous DNA end-joining

Non‐homologous end joining: Common interaction sites and exchange of multiple factors in the DNA repair process

Clinical variability and novel mutations in the NHEJ1 gene in patients with a Nijmegen breakage syndrome-like phenotype

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