Live cell imaging of XLF and XRCC4 reveals a novel view of protein assembly in the non-homologous end-joining pathway

Yano, Keiji; Chen, David J.

doi:10.4161/cc.7.10.5898

Cited by 68 publications

(58 citation statements)

References 44 publications

(66 reference statements)

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“…Moreover, the alignment of the paired ends within the synaptic complex is beneficial because of the reduced dimensionality of the search process (58). We speculate that the directionality of the alignment process may be mediated by the interaction of Ku with the opposite filament (19,59). When the DNA ends are properly positioned in a Butterfly structure, the filament will form a continuous bridge across the break.…”

Section: Discussionmentioning

confidence: 99%

“…Much of what we currently know about the temporal dynamics of NHEJ proteins in DSBs relies on time-resolved microscopy experiments using fluorescent-tagged NHEJ components (12,19,37,50); however, none of those studies addressed issues relating to positioning of the ends and the repair machinery during synapsis or the dynamics associated with the repair process before ligation. Specifically, it is not known how the broken ends are brought together, with respect to either their initial pairing configurations or the nature of the pairing interaction.…”

Section: Discussionmentioning

confidence: 99%

“…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)(16)(17)(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 DSBs (19)(20)(21)(22)(23)(24)(25). In addition, recent structural studies have shown that XRCC4 and XLF form filamentous structures in vitro (26)(27)(28).…”

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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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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

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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“…Biochemical evidence suggests that DNA-PKcs is required for the recruitment of the DNA ligase IV/XRCC4 complex to the chromatin after DNA damage (11). However, it has also been suggested that DNA-PKcs and the DNA ligase IV/XRCC4 complex are independently recruited to the DSB (12,13). In NHEJ, base pairing of DSB ends is essential, since ligation of blunt ends is not efficient (14).…”

Section: Introductionmentioning

confidence: 99%

Homologous Recombination Contributes to the Repair of DNA Double-Strand Breaks Induced by High-Energy Iron Ions

et al. 2010

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Zafar F., Seidler S.B., Kronenberg A., Schild D. and Wiese C. Homologous recombination contributes to the repair of DNA double-strand breaks induced by high-energy iron ions. Radiat. Res.To test the contribution of homologous recombinational repair (HRR) in repairing DNA damaged sites induced by high-energy iron ions, we used: 1) HRR-deficient rodent cells carrying a deletion in the RAD51D gene and 2) syngeneic human cells impaired for HRR by RAD51D or RAD51 knockdown using RNA interference. We show that in response to iron ions, HRR contributes to cell survival in rodent cells, and that HRR-deficiency abrogates RAD51 foci formation. Complementation of the HRR defect by human RAD51D rescues both enhanced cytotoxicity and RAD51 foci formation. For human cells irradiated with iron ions, cell survival is decreased, and, in p53 mutant cells, the levels of mutagenesis are increased when HRR is impaired. Human cells synchronized in S phase exhibit more pronounced resistance to iron ions as compared with cells in G1 phase, and this increase in radioresistance is diminished by RAD51 knockdown. These results implicate a role for RAD51-mediated DNA repair (i.e. HRR) in removing a fraction of clustered lesions induced by charged particle irradiation. Our results are the first to directly show the requirement for an intact HRR pathway in human cells in ensuring DNA repair and cell survival in response to high-energy high LET radiation.3

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“…By mathematical and numerical analysis and parameter estimation, the models are able to capture the qualitative biological features and show good agreement with experimental data. As conclusions, from the viewpoint of modeling, how the NHEJ proteins are recruited will be first discussed for connection between the classical sequential model [4] and recently proposed two-phase model [5]. Then how the NHEJ repair pathway is affected, by the length of DNA fragment [6], the complexity of DNA damage [7] and the chromatin structure [8], will be addressed.…”

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confidence: 99%

Modeling non-homologous end joining

Li¹,

Cucinotta

2011

Journal of Theoretical Biology

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Live cell imaging of XLF and XRCC4 reveals a novel view of protein assembly in the non-homologous end-joining pathway

Cited by 68 publications

References 44 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

Homologous Recombination Contributes to the Repair of DNA Double-Strand Breaks Induced by High-Energy Iron Ions

Modeling non-homologous end joining

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