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

Abstract

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

Ring finger protein 126 (RNF126) suppresses ionizing radiation–induced p53-binding protein 1 (53BP1) focus formation

“…Because the DDR has multiple pathways involved, there are several genome maintenance regulatory factors that play both positive and negative roles depending on the stage, or these pathways regulate each other ( 1 , 2 ). The first scenario concerning the role of RNF126 in DNA repair has been reported previously ( 28 , 29 ). In this study, we focused on the DNA damage response steps prior to repair.…”

“…In this study, we identified and characterized RNF126 as a novel negative regulator of RNF168-mediated ubiquitination and 53BP1 focus formation after DNA damage. Previous studies revealed the role of RNF126 as a positive regulator of DSB repair by controlling the expression level of BRCA1 in homologous recombination and by its ubiquitin ligase function in NHEJ ( 28 , 29 ). These reports highlighted the positive regulation of RNF126 in DSB repair, and our study elucidates a novel regulatory function of RNF126 in the DDR.…”

How cells ensure correct repair of DNA double-strand breaks

“…The effect of RNF138 ubiquitylation on Ku80 abundance at break sites appears to be independent of proteasome-mediated degradation. However, RNF8, SCF Fbxl12 , VCP-p97, and RNF126 have all been implicated in targeting Ku to the proteasome, and thereby shifting the balance of repair from NHEJ to HR (21)(22)(23)(24). The CRL4 E3 ubiquitin ligase, which contains either CUL4A or CUL4B, has additionally been implicated in removal of Ku via a ubiquitindependent mechanism that is also dependent on neddylation (25).…”

WWP2 ubiquitylates RNA polymerase II for DNA-PK-dependent transcription arrest and repair at DNA breaks

“…Ubiquitylated Ku70/Ku80 is then removed from chromatin in a VCP-dependent manner and targeted for degradation by the proteasome (van den Boom et al 2016). This allows for completion of NHEJ (Ishida et al 2017) or activation of end resection, thereby triggering the alternative DSB repair pathway of HR (Ismail et al 2015;van den Boom et al 2016). The FBXW7-associated cullin-RING ligase, on the other hand, regulates the recruitment of XRCC4 to DSB sites through its K63-linked ubiquitylation (Zhang et al 2016).…”