Impact of Amino Acid Substitutions in Two Functional Domains of Ku80: DNA-Damage-Sensing Ability of Ku80 and Survival after Irradiation

Koike, Manabu; Yutoku, Yasutomo; Koike, Aki

doi:10.1292/jvms.13-0283

Cited by 4 publications

(2 citation statements)

References 26 publications

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“…S3). Consistent with our finding that Ku80 and Ku70 each directly bind to RNF126 in vitro, mutation of the Ku70 binding site of Ku80 (A453H/V454H; HH) (11) or of the Ku80 binding site of Ku70 (L413R; LR) (12) did not affect the binding of either Ku protein to RNF126 ( Fig. 1D and E; Fig.…”

Section: Resultssupporting

confidence: 91%

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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Section: Resultssupporting

confidence: 91%

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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“…Additionally, NHEJ is an attractive target for strategies aimed at increasing the sensitivity of tumors to anticancer treatments including various radiotherapies. The regulation mechanism controlling the localization of core NHEJ factors, including Ku, plays a critical role in controlling NHEJ activity [11, 18]. Here, we demonstrated that EYFP-canine XRCC4 accumulation at micro-laser induced DSBs began immediately after irradiation.…”

Section: Discussionmentioning

confidence: 65%

Cloning, localization and focus formation at DNA damage sites of canine XRCC4

Koike

Yutoku

Koike

2016

The Journal of Veterinary Medical Science

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Various chemotherapies and radiation therapies are useful for killing cancer cells mainly by inducing DNA double-strand breaks (DSBs). Uncovering the molecular mechanisms of DSB repair processes is crucial for developing next-generation radiotherapies and chemotherapeutics for human and animal cancers. XRCC4 plays a critical role in Ku-dependent nonhomologous DNA-end joining (NHEJ) in human cells, and is one of the core NHEJ factors. The localization of core NHEJ factors, such as human Ku70 and Ku80, might play a crucial role in regulating NHEJ activity. Recently, companion animals, such as canines, have been proposed to be a good model in many aspects of cancer research. However, the localization and regulation mechanisms of core NHEJ factors in canine cells have not been elucidated. Here, we show that the expression and subcellular localization of canine XRCC4 changes dynamically during the cell cycle. Furthermore, EYFP-canine XRCC4 accumulates quickly at laser-microirradiated DSB sites. The structure of a putative human XRCC4 nuclear localization signal (NLS) is highly conserved in canine, chimpanzee and mouse XRCC4. However, the amino acid residue corresponding to the human XRCC4 K210, thought to be important for nuclear localization, is not conserved in canine XRCC4. Our findings might be useful for the study of the molecular mechanisms of Ku-dependent NHEJ in canine cells and the development of new radiosensitizers that target XRCC4.

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