Ku recruits XLF to DNA double‐strand breaks

Yano, Keiji; Morotomi-Yano, Keiko; Wang, Shih Ya; Uematsu, Naoya; Lee, Kyung Jong; Asaithamby, Aroumougame; Weterings, Eric; Chen, David J.

doi:10.1038/sj.embor.7401137

Cited by 160 publications

(181 citation statements)

References 25 publications

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“…Previously, live cell imaging studies had shown that the recruitment of XLF to DSBs is dependent upon Ku but not XRCC4 (27). Similarly, we found that the recruitment of Nej1 to an in vivo site-specific DSB measured by ChIP was dependent upon yKu but not Lif1 or Dnl4.…”

Section: Discussionsupporting

confidence: 58%

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Yeast Nej1 Is a Key Participant in the Initial End Binding and Final Ligation Steps of Nonhomologous End Joining

Chen¹,

Tomkinson

2011

Journal of Biological Chemistry

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In Saccharomyces cerevisiae, the key components of the nonhomologous end joining (NHEJ) pathway that repairs DNA double-strand breaks (DSBs) are yeast Ku (yKu), Mre11-Rad50-Xrs2, Dnl4-Lif1, and Nej1. Here, we examined the role of Nej1 in NHEJ by a combination of molecular genetic and biochemical approaches. As expected, the recruitment of Nej1 to in vivo DSBs is dependent upon yKu. Surprisingly, Nej1 is required for the stable binding of yKu to in vivo DSBs, in addition to Dnl4-Lif1. Thus, Nej1 and Dnl4-Lif1 are independently recruited by yKu to in vivo DSBs, forming a stable ternary complex that channels DSBs into the NHEJ pathway. In accord with these results, purified Nej1 interacts with yKu and preferentially binds to DNA ends bound by yKu. Furthermore, the binding of a mixture of Nej1 and Dnl4-Lif1 to DNA ends bound by yKu is greater than the sum of the binding of the individual proteins, indicating that pairwise interactions among yKu, Nej1, and Dnl4-Lif1 contribute to complex assembly at DNA ends. Nej1 stimulates intermolecular ligation by Dnl4-Lif1, but, more interestingly, the addition of Nej1 results in more than one intermolecular ligation per Dnl4 molecule. Thus, Nej1 not only plays an important role in determining repair pathway choice by participating in the initial NHEJ complex formed at DSBs but also contributes to the reactivation of Dnl4-Lif1 after repair is complete, thereby increasing the capacity of the NHEJ repair pathway.

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Section: Discussionsupporting

confidence: 58%

“…This notion is supported by parallel studies with the mammalian ortholog of Nej1, XLF (XRCC4-like factor; Cernunnos) (25)(26)(27). Notably, XLF not only stimulates the joining of cohesive DNA ends by DNA ligase IV-XRCC4 but also the joining of mismatched DNA ends (28 -34).…”

supporting

confidence: 56%

Yeast Nej1 Is a Key Participant in the Initial End Binding and Final Ligation Steps of Nonhomologous End Joining

Chen¹,

Tomkinson

2011

Journal of Biological Chemistry

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“…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).…”

mentioning

confidence: 99%

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.

157

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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“…The latter steps of NHEJ require additional proteins, including Artemis (endprocessing nuclease), XLF/Cerrunos, and the XRCC4/ligIV complex (ligase) (Jackson and Bartek 2009). More recent data on NHEJ assembly during DNA repair argue for a more complex model in which cooperative interactions between various NHEJ components orchestrate a precise architecture (Yano et al 2008). It has been shown that DNA-PK is autophosphorylated on DNA-PKcs at multiple residues, and such autophosphorylation is important for the completion of DNA repair (Meek et al 2008).…”

mentioning

confidence: 99%

Essential role for DNA-PK-mediated phosphorylation of NR4A nuclear orphan receptors in DNA double-strand break repair

et al. 2011

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DNA-dependent protein kinase (DNA-PK) is a central regulator of DNA double-strand break (DSB) repair; however, the identity of relevant DNA-PK substrates has remained elusive. NR4A nuclear orphan receptors function as sequence-specific DNA-binding transcription factors that participate in adaptive and stress-related cell responses. We show here that NR4A proteins interact with the DNA-PK catalytic subunit and, upon exposure to DNA damage, translocate to DSB foci by a mechanism requiring the activity of poly(ADP-ribose) polymerase-1 (PARP-1). At DNA repair foci, NR4A is phosphorylated by DNA-PK and promotes DSB repair. Notably, NR4A transcriptional activity is entirely dispensable in this function, and core components of the DNA repair machinery are not transcriptionally regulated by NR4A. Instead, NR4A functions directly at DNA repair sites by a process that requires phosphorylation by DNA-PK. Furthermore, a severe combined immunodeficiency (SCID)-causing mutation in the human gene encoding the DNA-PK catalytic subunit impairs the interaction and phosphorylation of NR4A at DSBs. Thus, NR4As represent an entirely novel component of DNA damage response and are substrates of DNA-PK in the process of DSB repair.

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Ku recruits XLF to DNA double‐strand breaks

Cited by 160 publications

References 25 publications

Yeast Nej1 Is a Key Participant in the Initial End Binding and Final Ligation Steps of Nonhomologous End Joining

Yeast Nej1 Is a Key Participant in the Initial End Binding and Final Ligation Steps of Nonhomologous End Joining

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

Essential role for DNA-PK-mediated phosphorylation of NR4A nuclear orphan receptors in DNA double-strand break repair

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