The E3 ligase RNF8 regulates KU80 removal and NHEJ repair.
ABSTRACT The ubiquitination cascade has a key role in the assembly of repair and signaling proteins at sites of double-strand DNA breaks. The E3 ubiquitin ligase RING finger protein 8 (RNF8) triggers the initial ubiquitination at double-strand DNA breaks, whereas sustained ubiquitination requires the downstream E3 ligase RING finger protein 168 (RNF168). It is not known whether RNF8 and RNF168 have discrete substrates and/or form different ubiquitin chains. Here we show that RNF168 acts with the ubiquitin-conjugating enzyme E2 13 (UBC13) and specifically synthesizes Lys63-linked chains, whereas RNF8 primarily forms Lys48-linked chains on chromatin, which promote substrate degradation. We also find that RNF8 regulates the abundance of the nonhomologous end-joining (NHEJ) repair protein KU80 at sites of DNA damage, and that RNF8 depletion results in prolonged retention of KU80 at damage sites and impaired nonhomologous end-joining repair. These findings reveal a distinct feature of RNF8 and indicate the involvement of the ubiquitination-mediated degradation pathway in DNA damage repair.
- SourceAvailable from: Hem Chandra Jha[Show abstract] [Hide abstract]
ABSTRACT: EBV latent antigen EBNA3C is implicated in B-cell immortalization and linked to several B-cell malignancies. Deregulation of H2AX can induce genomic instability with increased chromosomal aberrations which ultimately leads to tumorigenesis. Here we demonstrated that EBNA3C can attenuate H2AX expression at the transcript and protein levels. A reduction of total H2AX levels were clearly observed on infection of primary B-cells with wild type EBV, but not with EBNA3C knockout EBV recombinant virus. H2AX also interacted with EBNA3C through its N-terminal domain (residues 1-100). Furthermore, H2AX mutated at Ser139 failed to interact with EBNA3C. Luciferase based reporter assays also revealed that the binding domain of EBNA3C is sufficient for transcriptional inhibition of the H2AX promoter. EBNA3C also facilitated H2AX degradation through recruitment of components of the ubiquitin proteasome pathway. We further demonstrated that knockdown of H2AX in LCLs led to up-regulation of the Bub1 oncoprotein, and down-regulated expression of the p53. Overall, our study provides additional insights into EBV-associated B-cell lymphomas which is linked to regulation of the DNA damage response system in the infected cells.Importance I. EBNA3C down-regulates H2AX expression at the protein and transcript levels in epithelial cells, B-cells and EBV transformed LCLs.II. EBNA3C binds with wild type H2AX but not with the ser139 mutant of H2AX.III. The N-terminal (residue 1-100 amino acid) of EBNA3C is critical for binding to H2AX.IV. Localization of H2AX is predominantly nuclear in the presence of EBNA3C.V. H2AX knocked down in LCLs led to enhanced expression of Bub1 and downregulation of the tumor suppressor p53 both important for driving the oncogenic process.Journal of Virology 01/2014; · 5.08 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: The repair of DNA double strand breaks is essential for cell survival and several conserved pathways have evolved to ensure their rapid and efficient repair. The non-homologous end joining pathway is initiated when Ku binds to the DNA break site. Ku is an abundant nuclear heterodimer of Ku70 and Ku80 with a toroidal structure that allows the protein to slide over the broken DNA end and bind with high affinity. Once locked into placed, Ku acts as a tool-belt to recruit multiple interacting proteins, forming one or more non-homologous end joining complexes that act in a regulated manner to ensure efficient repair of DNA ends. Here we review the structure and functions of Ku and the proteins with which it interacts during non-homologous end joining.DNA repair 03/2014; · 4.20 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: DNA double-strand break (DSB) repair is crucial for the maintenance of genomic stability, and chromatin organization represents one important factor influencing repair efficiency. Mouse rod photoreceptors with their inverted heterochromatin organization containing a single large chromocenter in the middle of the nucleus provide a unique model system to study DSB repair in heterochromatin of living animals. We observed that adult rod photoreceptors repair only half of the induced DSBs within 1 day after damage induction, a defect that is neither observed in any other cell type of the adult retina nor in rod photoreceptor precursor cells of postnatal day 4 mice. We show that adult wild-type rods are deficient in a repair pathway involving ATM, a protein that promotes heterochromatic DSB repair by phosphorylating KAP1 and facilitating heterochromatin relaxation. Of note, we observed that rods fail to robustly accumulate active ATM at DSBs, exhibit low KAP1 levels, and display high levels of SPOC1, a factor suppressing KAP1 phosphorylation. Collectively, this results in dramatically reduced KAP1 phosphorylation and the inability to repair heterochromatic DSBs. Because the distinct heterochromatic structure of rods focuses transmitting light to enable vision at low photon levels, the inability to phosphorylate KAP1 and the failure to relax heterochromatin could serve to maintain this structure and the functionality of rods in the presence of DSBs. Collectively, our findings show that the unique chromatin organization of adult rods renders them incapable to efficiently repair heterochromatic DSBs, providing evidence that heterochromatin affects mammalian DSB repair in vivo.Current biology: CB 04/2014; · 10.99 Impact Factor