The RNF138 E3 ligase displaces Ku to promote DNA end resection and regulate DNA repair pathway choice

Ismail, Ismail Hassan; Gagné, Jean-Philippe; Genois, Marie-Michelle; Strickfaden, Hilmar; McDonald, Darin; Xu, Zhaochun; Poirier, Guy G.; Masson, Jean‐Yves; Hendzel, Michael J.

doi:10.1038/ncb3259

Cited by 119 publications

(137 citation statements)

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“…Similar results were also observed in cells depleted of RNF138 (Fig. 5B), an important player in directing DSBs to HR repair and promoting end resection (46,47). No changes in DDX1 protein levels were detected in either CtIP or RNF138 knockdown cells, indicating a cellular redistribution of DDX1 in these cells (Fig.…”

Section: Ddx1 Contributes To Dsb Repair and Cell Survival Post-irsupporting

confidence: 72%

DEAD Box 1 Facilitates Removal of RNA and Homologous Recombination at DNA Double-Strand Breaks

Germain

Poon

et al. 2016

Molecular and Cellular Biology

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Although RNA and RNA-binding proteins have been linked to double-strand breaks (DSBs), little is known regarding their roles in the cellular response to DSBs and, if any, in the repair process. Here, we provide direct evidence for the presence of RNA-DNA hybrids at DSBs and suggest that binding of RNA to DNA at DSBs may impact repair efficiency. Our data indicate that the RNAunwinding protein DEAD box 1 (DDX1) is required for efficient DSB repair and cell survival after ionizing radiation (IR), with depletion of DDX1 resulting in reduced DSB repair by homologous recombination (HR). While DDX1 is not essential for end resection, a key step in homology-directed DSB repair, DDX1 is required for maintenance of the single-stranded DNA once generated by end resection. We show that transcription deregulation has a significant effect on DSB repair by HR in DDX1-depleted cells and that RNA-DNA duplexes are elevated at DSBs in DDX1-depleted cells. Based on our combined data, we propose a role for DDX1 in resolving RNA-DNA structures that accumulate at DSBs located at sites of active transcription. Our findings point to a previously uncharacterized requirement for clearing RNA at DSBs for efficient repair by HR. DEAD box proteins are a family of putative RNA helicases that function by altering RNA secondary structure. This protein family has been implicated in all aspects of RNA metabolism. The DEAD box 1 gene (DDX1) is a widely expressed gene that is misexpressed in a number of cancers, including retinoblastoma, neuroblastoma, and breast cancer (1, 2). Knockout of DDX1 leads to early embryonic lethality in mice and severely reduced fertility in flies (3, 4). DDX1 is involved in the transport of RNAs from the nucleus to the cytoplasm and regulates cytoplasmic localization of the splicing-regulatory protein KSRP (5). In neurons, DDX1 resides in RNA-transporting granules, cytoplasmic organelles that regulate the localization and expression of target mRNAs (6, 7). DDX1 has also been identified as a core subunit of the human tRNA ligase complex which is essential for tRNA splicing (8).In addition to its roles in RNA metabolism, DDX1 has been implicated in the cellular response to DNA double-strand breaks (DSBs). Upon treatment of cells with ionizing radiation (IR), DDX1 rapidly accumulates at a subset of DNA DSBs (ϳ30%), where it forms IR-induced foci that colocalize with ␥-H2AX, a marker for DSBs (9). DDX1 coimmunoprecipitates with the MRN (MRE11-RAD50-NBS1) complex, the early sensor of DNA DSBs, and ATM (ataxia telangiectasia mutated) protein, the key transducer of the signaling cascade in response to DSBs (10, 11). DSBs induce DDX1 phosphorylation in an ATM-dependent manner. Notably, IR-induced DDX1 foci are lost when cells are treated with RNase H, an enzyme that specifically digests RNA from RNA-DNA hybrids (9). These results suggest that RNA-DNA double-stranded structures are required for the presence and/or retention of DDX1 at DSBs. In line with this observation, biochemical analysis has shown that DDX1 can unwind both...

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Section: Ddx1 Contributes To Dsb Repair and Cell Survival Post-irsupporting

confidence: 72%

DEAD Box 1 Facilitates Removal of RNA and Homologous Recombination at DNA Double-Strand Breaks

Germain

Poon

et al. 2016

Molecular and Cellular Biology

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133

138

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“…CDKs promote these 2 steps in the process of DNA resection by phosphorylating CtIP, NBS1, or EXO1, respectively (14,55). Ku ubiquitination mediated by the E3 ligase RNF138 and subsequent degradation promote DNA resection (57). Intriguingly, BRAC1 promotes, whereas RIF1 acts as the effector of 53BP1 to inhibit, DNA resection and HR (55,56).…”

Section: Methodsmentioning

confidence: 99%

“…CDKs, RNF138, chromatin remodeling factors, and others (14,(54)(55)(56)(57). Initiation of DNA end resection by the MRN complex and CtIP removes Ku from DNA ends to generate ssDNA overhangs that not only inhibit NHEJ but also provide a platform to recruit proteins involved in HR repair (47).…”

Section: Methodsmentioning

confidence: 99%

Targeting Mcl-1 enhances DNA replication stress sensitivity to cancer therapy

Guo¹,

Magis²,

Xu³

et al. 2017

Journal of Clinical Investigation

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“…Instead, RNF8-and NEDD8-dependent ubiquitin ligases have been found to mediate Ku80 and Ku70 ubiquitylation, respectively, in mammalian cells (7,8). Furthermore, RNF138 was shown to ubiquitylate Ku80 at S-G 2 phases of the cell cycle (6). However, it has remained unclear whether these are the only ubiquitin ligases that target the Ku heterodimer and which residues of Ku80 and Ku70 are ubiquitylated, with the exception of a few sites whose mutation does not affect Ku release from damaged DNA in chromatin (8).…”

mentioning

confidence: 99%

“…SCF Fbxl12 mediates ubiquitylation of Ku80 in Xenopus eggs (5), but this mechanism is not likely conserved in mammalian cells (6). Instead, RNF8-and NEDD8-dependent ubiquitin ligases have been found to mediate Ku80 and Ku70 ubiquitylation, respectively, in mammalian cells (7,8).…”

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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The RNF138 E3 ligase displaces Ku to promote DNA end resection and regulate DNA repair pathway choice

Cited by 119 publications

References 49 publications

DEAD Box 1 Facilitates Removal of RNA and Homologous Recombination at DNA Double-Strand Breaks

DEAD Box 1 Facilitates Removal of RNA and Homologous Recombination at DNA Double-Strand Breaks

Targeting Mcl-1 enhances DNA replication stress sensitivity to cancer therapy

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

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