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

Supplemental material for this article may be found at https://doi.org/10.1128/MCB.00347-16.

ACKNOWLEDGMENTS

We thank H. Ogiwara and T. Kohno for providing the H1299dA3-1#1 cell line; M. Jasin and H. Tauchi for providing the plasmid pCMV-3xNLS-I-SceI; H. Miyoshi for providing the plasmids pCAG-HIVgp and pCMV-VSV-G-RSV-Rev; K. I. Nakayama for providing the plasmids pCIneo-His₆-T7-Ub and pGEX-6P-Ub; H. Nakatsumi for providing the plasmid CSII-EF-IRES-puro; L. Li, S. Nakajima, R. Watanabe, and N. Ishii for technical assistance with DNA damage experiments; K. Murayama for help with structural analysis of the Ku70-Ku80 complex; T. Kitamura for providing pMX-puro and Plat-E cells; the Biomedical Research Core of Tohoku University Graduate School of Medicine for technical support; and other laboratory members for discussion.

We have no conflicts of interest to declare.

This work was supported by grants 20570175 and 25891003 from the Japan Society for the Promotion of Science.