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

Ropars, Virginie; Drevet, Pascal; Legrand, Pierre; Baconnais, Sonia; Amram, Jeremy; Faure, Guilhem; Márquez, José Antonio; Piétrement, Olivier; Guérois, Raphaël; Callebaut, Isabelle; Cam, Eric Le; Revy, Patrick; Villartay, Jean‐Pierre de; Charbonnier, Jean‐Baptiste

doi:10.1073/pnas.1100758108

Cited by 123 publications

(140 citation statements)

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“…previous reports that XLF/XRCC4 can form filaments in the absence of DNA (41). When the proteins Ku, LX, and XLF were reacted, we observed similar filaments when staining for LigIV ( Fig.…”

Section: Sr Imagingsupporting

confidence: 82%

“…have shown that XLF and XRCC4 form extended filaments (26,27,(38)(39)(40)(41)(42). Importantly, our SR imaging revealed that XRCC4 and XLF filaments exist in vivo, and also that LigIV appears to form filamentous structures in cells (Fig.…”

Section: Sr Imagingsupporting

confidence: 52%

“…The presence of filaments at either side of the break is advantageous, allowing for more pairing configurations compared with pairing events occurring only at the ends, thereby increasing the probability of pairing. When two Caterpillar structures meet, the XRCC4/XLF/LigIV filaments pair in either a direct end-to-end or an adjacent configuration, forming Butterfly structures (41,42).…”

Section: Discussionmentioning

confidence: 99%

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Organization and dynamics of the nonhomologous end-joining machinery during DNA double-strand break repair

Reid

Keegan

Leo‐Macias

et al. 2015

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

154

168

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Nonhomologous end-joining (NHEJ) is a major repair pathway for DNA double-strand breaks (DSBs), involving synapsis and ligation of the broken strands. We describe the use of in vivo and in vitro single-molecule methods to define the organization and interaction of NHEJ repair proteins at DSB ends. Super-resolution fluorescence microscopy allowed the precise visualization of XRCC4, XLF, and DNA ligase IV filaments adjacent to DSBs, which bridge the broken chromosome and direct rejoining. We show, by singlemolecule FRET analysis of the Ku/XRCC4/XLF/DNA ligase IV NHEJ ligation complex, that end-to-end synapsis involves a dynamic positioning of the two ends relative to one another. Our observations form the basis of a new model for NHEJ that describes the mechanism whereby filament-forming proteins bridge DNA DSBs in vivo. In this scheme, the filaments at either end of the DSB interact dynamically to achieve optimal configuration and end-to-end positioning and ligation.genomic integrity | DNA repair | nonhomologous end-joining | super-resolution microscopy | single-molecule FRET C hromosomal double-strand breaks (DSBs), considered the most cytotoxic form of DNA damage, occur as a result of normal cellular processes (1, 2) as well as cancer therapies (3-5). The cellular DNA damage response (DDR) and repair pathways responsible for maintaining genomic integrity are highly regulated and synchronized processes, both temporally and spatially, involving the coordinated recruitment, assembly, and disassembly of numerous macromolecular complexes (6, 7). In mammalian cells, nonhomologous end-joining (NHEJ) is the primary DSB repair pathway; it is active throughout the cell cycle and is crucial for viability. Dysfunctional NHEJ is associated with several clinical conditions, including LIG4 syndrome and severe combined immunodeficiency (1,8). Despite its importance, however, the details of how the NHEJ complex assembles at DSBs, brings together a pair of breaks, and organizes subsequent catalytic repair steps remain unknown.In NHEJ, DSBs are initially recognized by the Ku 70/80 heterodimer (Ku), which encircles dsDNA ends (Ku:DNA) and serves as a molecular scaffold for recruitment of DNA-dependent protein kinase catalytic subunit (DNA-PKcs), XRCC4 (X-ray repair cross-complementing protein 4), XLF (XRCC4 like factor), and DNA ligase IV (LigIV) (1,(9)(10)(11)(12)(13)(14). Previous NHEJ models suggested that after binding of Ku to DNA ends, DNA-PKcs binds Ku:DNA to form a DNA-PK holoenzyme and bridges the broken ends (15-18); however, recent experiments indicate that DNAPKcs may have different roles in NHEJ, such as the stabilization of core NHEJ factors, recruitment and retention of accessory factors, involvement in the DDR signaling cascade, and repair of complex and clustered . In addition, recent structural studies have shown that XRCC4 and XLF form filamentous structures in vitro (26-28). Whether such filaments mediate repair in vivo has not yet been determined.Our present understanding of the cellular NHEJ response to DSBs ...

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“…previous reports that XLF/XRCC4 can form filaments in the absence of DNA (41). When the proteins Ku, LX, and XLF were reacted, we observed similar filaments when staining for LigIV ( Fig.…”

Section: Sr Imagingsupporting

confidence: 82%

Section: Sr Imagingsupporting

confidence: 52%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Organization and dynamics of the nonhomologous end-joining machinery during DNA double-strand break repair

Reid

Keegan

Leo‐Macias

et al. 2015

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

154

168

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“…Accordingly, Cernu Ϫ/Ϫ mice crossed onto the P53 KO background did not develop B cell lymphoma, as opposed to the case with other NHEJ deficiency situations (26). We propose a speculative yet plausible explanation for this intriguing observation, based on our recent report on the Cernunnos-Xrcc4 complex structure (27). Crystals of Cernunnos-Xrcc4 complexes revealed that both homodimers associate with each other in long filaments through their head domains, helping to tether the broken DNA ends by creating a "DNA ligation synapse" (27)(28)(29)(30).…”

Section: Discussionmentioning

confidence: 60%

Cernunnos Deficiency Reduces Thymocyte Life Span and Alters the T Cell Repertoire in Mice and Humans

Vera

Rivera-Munoz

Abramowski

et al. 2013

Molecular and Cellular Biology

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128

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f Cernunnos is a DNA repair factor of the nonhomologous end-joining machinery. Its deficiency in humans causes radiosensitive severe combined immune deficiency (SCID) with microcephaly, characterized in part by a profound lymphopenia. In contrast to the human condition, the immune system of Cernunnos knockout (KO) mice is not overwhelmingly affected. In particular, Cernunnos is dispensable during V(D)J recombination in lymphoid cells. Nevertheless, the viability of thymocytes is reduced in Cernunnos KO mice, owing to the chronic activation of a P53-dependent DNA damage response. This translates into a qualitative alteration of the T cell repertoire to one in which the most distal V␣ and J␣ segments are missing. This results in the contraction of discrete T cell populations, such as invariant natural killer T (iNKT) and mucosa-associated invariant T (MAIT) cells, in both humans and mice.T he immune system is the site of intense genome dynamics, in particular during the development and maturation of B and T lymphocytes in bone marrow and the thymus, when antigen receptor genes are rearranged through V(D)J recombination prior to their expression. DNA damages are also likely to occur during the several phases of intense proliferation which accompany the development of B and T cells.V(D)J recombination is the prototypical example of the generation of a programmed DNA double-strand break (DNA-dsb) during lymphoid development through the activity of recombination activating genes 1 and 2 (Rag1/2) on immunoglobulin (Ig) and T cell receptor (TCR) genes (see the work of Helmink and Sleckman [1] for a recent review). The resulting DNA-dsb is resolved by the nonhomologous end-joining (NHEJ) DNA repair pathway, composed of seven core components (see the work of Lieber [2] for a recent review). The Cernunnos-Xrcc4-DNA ligase IV complex ultimately reseals the DNA-dsb. Cernunnos, also known as Xrcc4-like factor (XLF), was the last NHEJ factor that was independently identified, through a survey of RS-SCID patients (3) and a yeast two-hybrid screen with Xrcc4 as a bait (4). Cernunnos and Xrcc4 adopt the same overall three-dimensional crystal structure (5, 6) and, together with DNA ligase IV, are parts of the same complex (4, 7). Cernunnos stimulates the DNA-joining activity of the Xrcc4-DNA ligase IV complex (8, 9). V(D)J recombination constitutes a central checkpoint in the development of the immune system, as its defect leads to abortive B and T cell maturation in vivo, resulting in severe combined immune deficiency (SCID) (10), but its first recognized function was the generation of a diverse antigenic repertoire through the combinatorial association of variable, diversity, and joining segments that encode the variable domains of both Ig and TCRs (11). Numerous examples show that a reduced V(D)J recombinase activity affects the extent of antigenic diversity of immune receptors in mice and humans. The resulting immune deregulation may then lead to autoimmunity, increased susceptibility to infections, or the development of vario...

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“…Since its discovery in 2005, several functions have been described for XLF. It stimulates LIG4 activity, 21,22 and it can form long filaments with XRCC4 that keep the DNA ends together in a ligation synapse, 24,[48][49][50][51] and XLF is essential for gap-filling by polymerase (pol) l and polm during NHEJ. The latter function is interesting, as poll and polm belong to the same polX family of polymerases as For personal use only.…”

Section: Discussionmentioning

confidence: 99%

XLF deficiency results in reduced N-nucleotide addition during V(D)J recombination

IJspeert

Rozmus

Schwarz

et al. 2016

Blood

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Key Points• XLF belongs to the NHEJ ligation complex and has a dual role in DNA doublestrand break repair and V(D)J recombination.• XLF is involved in Nnucleotide addition, and thereby contributes to junctional diversity of the antigen receptors.Repair of DNA double-strand breaks (DSBs) by the nonhomologous end-joining pathway (NHEJ) is important not only for repair of spontaneous breaks but also for breaks induced in developing lymphocytes during V(D)J (variable [V], diversity [D], and joining [J] genes) recombination of their antigen receptor loci to create a diverse repertoire. Mutations in the NHEJ factor XLF result in extreme sensitivity for ionizing radiation, microcephaly, and growth retardation comparable to mutations in LIG4 and XRCC4, which together form the NHEJ ligation complex. However, the effect on the immune system is variable (mild to severe immunodeficiency) and less prominent than that seen in deficiencies of NHEJ factors ARTEMIS and DNA-dependent protein kinase catalytic subunit, with defects in the hairpin opening step, which is crucial and unique for V(D)J recombination. Therefore, we aimed to study the role of XLF during V(D)J recombination. We obtained clinical data from 9 XLF-deficient patients and performed immune phenotyping and antigen receptor repertoire analysis of immunoglobulin (Ig) and T-cell receptor (TR) rearrangements, using next-generation sequencing in 6 patients. The results were compared with XRCC4 and LIG4 deficiency. Both Ig and TR rearrangements showed a significant decrease in the number of nontemplated (N) nucleotides inserted by terminal deoxynucleotidyl transferase, which resulted in a decrease of 2 to 3 amino acids in the CDR3. Such a reduction in the number of N-nucleotides has a great effect on the junctional diversity, and thereby on the total diversity of the Ig and TR repertoire. This shows that XLF has an important role during V(D)J recombination in creating diversity of the repertoire by stimulating N-nucleotide insertion. (Blood. 2016;128(5):650-659)

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

Cited by 123 publications

References 38 publications

Organization and dynamics of the nonhomologous end-joining machinery during DNA double-strand break repair

Organization and dynamics of the nonhomologous end-joining machinery during DNA double-strand break repair

Cernunnos Deficiency Reduces Thymocyte Life Span and Alters the T Cell Repertoire in Mice and Humans

XLF deficiency results in reduced N-nucleotide addition during V(D)J recombination

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