Ku80 removal from DNA through double strand break–induced ubiquitylation

Postow, Lisa; Ghenoiu, Cristina; Woo, Eileen M.; Krutchinsky, Andrew N.; Chait, Brian T.; Funabiki, Hironori

doi:10.1083/jcb.200802146

Cited by 132 publications

(150 citation statements)

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“…Xenopus egg extracts, removal of Ku70/80 from DNA is dependent on Ku80 ubiquitylation, which occurs after loading of the heterodimer onto chromatin and induces not only the release of Ku80 from DNA but also its degradation by the proteasome (4). SCF Fbxl12 mediates ubiquitylation of Ku80 in Xenopus eggs (5), but this mechanism is not likely conserved in mammalian cells (6).…”

mentioning

confidence: 99%

Ubiquitylation of Ku80 by RNF126 Promotes Completion of Nonhomologous End Joining-Mediated DNA Repair

Ishida

Nakagawa

Iemura

et al. 2017

Molecular and Cellular Biology

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Repair of damaged DNA is critical for maintenance of genetic information. In eukaryotes, DNA double-strand breaks (DSBs) are recognized by the Ku70-Ku80 heterodimer, which then recruits proteins that mediate repair by nonhomologous end joining (NHEJ). Prolonged retention of Ku70/80 at DSBs prevents completion of repair, however, with ubiquitylation of Ku80 having been implicated in Ku70/80 dissociation from DNA. Here, we identify RNF126 as a ubiquitin ligase that is recruited to DSBs and ubiquitylates Ku80, with UBE2D3 serving as an E2 enzyme. Knockdown of RNF126 prevented Ku70/80 dissociation from DSBs and inhibited break repair. Attenuation of Ku80 ubiquitylation by replacement of ubiquitylation site lysines with arginine residues delayed Ku70/80 release from chromatin after DSB induction by genotoxic insults. Together, our data indicate that RNF126 is a novel regulator of NHEJ that promotes completion of DNA repair by ubiquitylating Ku80 and releasing Ku70/80 from damaged DNA.KEYWORDS DNA repair, Ku80, RNF126, nonhomologous end joining (NHEJ), ubiquitylation D NA damage poses a threat to living organisms because they rely on the genetic code to execute cellular functions. Genomic integrity is maintained in the face of DNA damage by the operation of dedicated DNA repair machinery. In eukaryotic cells, DNA double-strand breaks (DSBs), which constitute the most deleterious type of DNA damage, are repaired by two major, mechanistically distinct pathways: homologous recombination (HR) and nonhomologous end joining (NHEJ). HR replaces lost or damaged DNA sequence with high fidelity in a manner dependent on the presence of an intact sister chromatid as a template. HR thus functions only during S and G 2 phases of the cell cycle, when a sister chromatid is available (1). In contrast, NHEJ is active throughout the cell cycle and repairs DSBs by directly ligating chromosome ends without the use of a DNA template. As a consequence of its mode of action, however, NHEJ is error prone (2).The NHEJ pathway is initiated on recognition of DSBs by chromatin-remodeling factors and the Ku70-Ku80 heterodimer, the latter of which recruits additional factors to process the DSBs for repair. The Ku heterodimer forms a ring-shaped toroidal structure, through which the broken end of DNA is threaded. Given its abundance and the high affinity of Ku70/80 for DNA ends, a specific mechanism is thought to be required to dislodge the heterodimer from DNA after the recruitment of repair proteins in order to prevent its inhibition of repair completion and postrepair recovery (3). In

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confidence: 99%

Ubiquitylation of Ku80 by RNF126 Promotes Completion of Nonhomologous End Joining-Mediated DNA Repair

Ishida

Nakagawa

Iemura

et al. 2017

Molecular and Cellular Biology

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“…In addition, Ku also has been shown to be posttranslationally modified in a regulated manner (Douglas et al, 2005;Postow et al, 2008). Thus, it is possible that the interaction between Ku and PARP-1 is enhanced by the post-translational modification of one or both factors that occurs subsequent to bleomycin.…”

Section: Discussionmentioning

confidence: 99%

Activation of PARP-1 in response to bleomycin depends on the Ku antigen and protein phosphatase 5

Fang¹,

Soubeyrand²,

Haché

2010

Oncogene

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Poly (ADP-ribose) polymerase-1 (PARP-1) has an important role in the cellular response to a broad spectrum of DNA lesions. PARP-1 is strongly activated in response to double-stranded DNA breaks (DSBs), yet its contribution to the DSB response is poorly understood. Here we used bleomycin, a radiomimetic that generates DSBs with high specificity to focus on the response of PARP-1 to DSBs. We report that the induction of PARP-1 activity by bleomycin depends on the Ku antigen, a nonhomologous-DNA-End-Joining factor and protein phosphatase 5 (PP5). PARP-1 activation in response to bleomycin was reduced over 10-fold in Ku-deficient cells, whereas its activation in response to U.V. was unaffected. PARP-1 activation was rescued by reexpression of Ku, but was refractory to manipulation of DNA-dependent protein kinase or ATM. Similarly, PARP-1 activation subsequent to bleomycin was reduced 2-fold on ablation of PP5 and was increased 5-fold when PP5 was overexpressed. PP5 seemed to act directly on PARP-1, as its basal phosphorylation was reduced on overexpression of PP5, and PP5 dephosphorylated PARP-1 in vitro. These results highlight the functional importance of Ku antigen and PP5 for PARP-1 activity subsequent to DSBs.

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“…Two of the key NHEJ enzymes, Ku70 and Ku80, are susceptible to ubiquitination (Gama et al, 2006). Ku70 and Ku 80 are degraded by the proteasome, and MG132 delays proteolysis of these proteins (Postow et al, 2008;Enokido et al, 2010). Ubiquitinated Ku70 in human cells has been detected in the absence of proteasome inhibitors, indicating that this modification may not always serve as a degradation signal for Ku70; yet, apoptotic stress does upregulate degradative Ku70 ubiquitination (Gama et al, 2006).…”

Section: Double-strand Break Repair and The Upsmentioning

confidence: 99%

“…Ku70 and Ku80 stabilize one another, but ubiquitinated Ku70 appears to inhibit Ku70/Ku80 complex formation (Gama et al, 2006). Ku80 is polyubiquitinated when bound to DSBs in Xenopus laevis egg extracts, and this K48-linked polyubiquitination is related to removal of Ku80 from DNA (Postow et al, 2008). However, polyubiquitination-induced removal of Ku80 from DSBs is not required for the completion of NHEJ (Postow et al, 2008).…”

Section: Double-strand Break Repair and The Upsmentioning

confidence: 99%

“…Ku80 is polyubiquitinated when bound to DSBs in Xenopus laevis egg extracts, and this K48-linked polyubiquitination is related to removal of Ku80 from DNA (Postow et al, 2008). However, polyubiquitination-induced removal of Ku80 from DSBs is not required for the completion of NHEJ (Postow et al, 2008). In yeast, aberrant expression of key proteosome regulating transcription factor Rpn4 appears to hinder NHEJ but not HR (Ju et al, 2010).…”

Section: Double-strand Break Repair and The Upsmentioning

confidence: 99%

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