LRIK interacts with the Ku70-Ku80 heterodimer enhancing the efficiency of NHEJ repair.
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ABSTRACT:
SUBMITTER: Wang D
PROVIDER: S-EPMC7852670 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Androgens are known to protect prostate cancer cells from DNA damage. Recent studies showed regulation of DNA repair genes by androgen receptor signaling in prostate cancers. ELL-associated factor 2 (EAF2) is an androgen-regulated tumor suppressor and its intracellular localization can be modulated by ultraviolet light, suggesting a potential role for EAF2 in androgen regulation of DNA repair in prostate cancer cells. Here we show that knockdown of EAF2 or its homolog EAF1 sensitized prostate cancer cells to DNA damage and the sensitization did not require p53. EAF2 knockout mouse prostate was also sensitized to γ-irradiation. Furthermore, EAF2 knockdown blocked androgen repression of LNCaP or C4-2 cells from doxorubicin induction of γH2ax, a DNA damage marker. In human prostate cancer specimens, EAF2 expression was inversely correlated with the level of γH2ax. Further analysis showed that EAF2 and EAF1 are required for the recruitment and retention of Ku70/Ku80 to DNA damage sites and play a functional role in nonhomologous end-joining DNA repair. These findings provide evidence for EAF2 as a key factor mediating androgen protection of DNA damage via Ku70/Ku80 in prostate cancer cells.
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Project description: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.
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Project description:Recognition of DNA double-strand breaks during non-homologous end joining is carried out by the Ku70-Ku80 protein, a 150 kDa heterodimer that recruits the DNA repair kinase DNA-dependent protein kinase catalytic subunit (DNA-PKcs) to the lesion. The atomic structure of a truncated Ku70-Ku80 was determined; however, the subunit-specific carboxy-terminal domain of Ku80--essential for binding to DNA-PKcs--was determined only in isolation, and the C-terminal domain of Ku70 was not resolved in its DNA-bound conformation. Both regions are conserved and mediate protein-protein interactions specific to mammals. Here, we reconstruct the three-dimensional structure of the human full-length Ku70-Ku80 dimer at 25 A resolution, alone and in complex with DNA, by using single-particle electron microscopy. We map the C-terminal regions of both subunits, and their conformational changes after DNA and DNA-PKcs binding to define a molecular model of the functions of these domains during DNA repair in the context of full-length Ku70-Ku80 protein.
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Project description:Double-strand breaks (DSBs) are repaired through two major pathways, homology-directed recombination (HDR) and non-homologous end joining (NHEJ). The choice between these two pathways is largely influenced by cell cycle phases. HDR can occur only in S/G2 when sister chromatid can provide homologous templates, whereas NHEJ can take place in all phases of the cell cycle except mitosis. Central to NHEJ repair is the Ku70/80 heterodimer which forms a ring structure that binds DSB ends and serves as a platform to recruit factors involved in NHEJ. Upon completion of NHEJ repair, DNA double strand-encircling Ku dimers have to be removed. The removal depends on ubiquitylation and proteasomal degradation of Ku80 by the ubiquitin E3 ligases RNF8. Here we report that RNF8 is a substrate of APCCdh1 and the latter keeps RNF8 level in check at DSBs to prevent premature turnover of Ku80.
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Project description:IK is a mitotic factor that promotes cell cycle progression. Our previous investigation of 271 endometrial cancer (EC) samples from the Cancer Genome Atlas (TCGA) dataset showed IK somatic mutations were enriched in a cluster of patients with high-grade and high-stage cancers, and this group had longer survival. This study provides insight into how IK somatic mutations contribute to EC pathophysiology. We analyzed the somatic mutational landscape of IK gene in 547 EC patients using expanded TCGA dataset. Co-immunoprecipitation and mass spectrometry were used to identify protein interactions. In vitro and in vivo experiments were used to evaluate IK's role in EC. The patients with IK-inactivating mutations had longer survival during 10-year follow-up. Frameshift and stop-gain were common mutations and were associated with decreased IK expression. IK knockdown led to enrichment of G2/M phase cells, inactivation of DNA repair signaling mediated by heterodimerization of Ku80 and Ku70, and sensitization of EC cells to cisplatin treatment. IK/Ku80 mutations were accompanied by higher mutation rates and associated with significantly better overall survival. Inactivating mutations of IK gene and loss of IK protein expression were associated with weakened Ku80/Ku70-mediated DNA repair, increased mutation burden, and better response to chemotherapy in patients with EC.
| S-EPMC8160817 | biostudies-literature
Project description:Multiple DNA repair pathways may be involved in the removal of the same DNA lesion caused by endogenous or exogenous agents. Although distinct DNA repair machinery fulfill overlapping roles in the repair of DNA lesions, the mechanisms coordinating different pathways have not been investigated in detail. Here, we show that Ku70, a core protein of nonhomologous end-joining (NHEJ) repair pathway, can directly interact with DNA polymerase-β (Pol-β), a central player in the DNA base excision repair (BER), and this physical complex not only promotes the polymerase activity of Pol-β and BER efficiency but also enhances the classic NHEJ repair. Moreover, we find that DNA damages caused by methyl methanesulfonate (MMS) or etoposide promote the formation of Ku70-Pol-β complexes at the repair foci. Furthermore, suppression of endogenous Ku70 expression by small interfering RNA reduces BER efficiency and leads to higher sensitivity to MMS and accumulation of the DNA strand breaks. Similarly, Pol-β knockdown impairs total-NHEJ capacity but only has a slight influence on alternative NHEJ. These results suggest that Pol-β and Ku70 coordinate 2-way crosstalk between the BER and NHEJ pathways.-Xia, W., Ci, S., Li, M., Wang, M., Dianov, G. L., Ma, Z., Li, L., Hua, K., Alagamuthu, K. K., Qing, L., Luo, L., Edick, A. M., Liu, L., Hu, Z., He, L., Pan, F., Guo, Z. Two-way crosstalk between BER and c-NHEJ repair pathway is mediated by Pol-β and Ku70.
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Project description:BackgroundHIV-1 integration results in genomic DNA gaps that are repaired by cellular DNA repair pathways. This step of the lentiviral life cycle remains poorly understood despite its crucial importance for successful replication. We and others reported that Ku70 protein of the non-homologous end joining pathway (NHEJ) directly binds HIV-1 integrase (IN). Here, we studied the importance of this interaction for post-integrational gap repair and the recruitment of NHEJ factors in this process.ResultsWe engineered HIV-based pseudovirus with mutant IN defective in Ku70 binding and generated heterozygous Ku70, Ku80 and DNA-PKcs human knockout (KO) cells using CRISPR/Cas9. KO of either of these proteins or inhibition of DNA-PKcs catalytic activity substantially decreased the infectivity of HIV-1 with native IN but not with the mutant one. We used a recently developed qPCR assay for the measurement of gap repair efficiency to show that HIV-1 with mutant IN was defective in DNA post-integrational repair, whereas the wild type virus displayed such a defect only when NHEJ system was disrupted in any way. This effect was present in CRISPR/Cas9 modified 293T cells, in Jurkat and CEM lymphoid lines and in primary human PBMCs.ConclusionsOur data provide evidence that IN recruits DNA-PK to the site of HIV-1 post-integrational repair due to Ku70 binding-a novel finding that explains the involvement of DNA-PK despite the absence of free double stranded DNA breaks. In addition, our data clearly indicate the importance of interactions between HIV-1 IN and Ku70 in HIV-1 replication at the post-integrational repair step.
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Project description:We report the Ku80/Ku70 complex associated RNA in HEK293T cells using RNA-seq following PAR-Clip.
2024-04-09 | GSE219220 | GEO
Project description:The Ku70/80 heterodimer binds to DNA ends and attracts other proteins involved in the non-homologous end-joining (NHEJ) pathway of DNA double-strand break repair. We developed a novel assay to measure DNA binding and release kinetics using differences in Förster resonance energy transfer (FRET) of the ECFP-Ku70/EYFP-Ku80 heterodimer in soluble and DNA end bound states. We confirmed that the relative binding efficiencies of various DNA substrates (blunt, 3 nucleotide 5' extension, and DNA hairpin) measured in the FRET assay reflected affinities obtained from direct measurements using surface plasmon resonance. The FRET assay was subsequently used to investigate Ku70/80 behavior in the context of a DNA-dependent kinase (DNA-PK) holocomplex. As expected, this complex was much more stable than Ku70/80 alone, and its stability was influenced by DNA-PK phosphorylation status. Interestingly, the Ku80 C-terminal extension contributed to DNA-PK complex stability but was not absolutely required for its formation. The Ku70 C-terminal SAP domain, on the other hand, was required for the stable association of Ku70/80 to DNA ends, but this effect was abrogated in DNA-PK holocomplexes. We conclude that FRET measurements can be used to determine Ku70/80 binding kinetics. The ability to do this in complex mixtures makes this assay particularly useful to study larger NHEJ protein complexes on DNA ends.
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Project description:Reduced NME1 expression in melanoma cell lines, mouse models of melanoma, and melanoma specimens in human patients is associated with increased metastatic activity. Herein, we investigate the role of NME1 in repair of double-stranded breaks (DSBs) and choice of double-strand break repair (DSBR) pathways in melanoma cells. Using chromatin immunoprecipitation, NME1 was shown to be recruited rapidly and directly to DSBs generated by the homing endonuclease I-PpoI. NME1 was recruited to DSBs within 30 min, in concert with recruitment of ataxia-telangiectasia mutated (ATM) protein, an early step in DSBR complex formation, as well as loss of histone 2B. NME1 was detected up to 5 kb from the break site after DSB induction, suggesting a role in extending chromatin reorganization away from the repair site. shRNA-mediated silencing of NME1 expression led to increases in the homologous recombination (HR) and non-homologous end-joining (NHEJ) pathways of double-strand break repair (DSBR), and reduction in the low fidelity, alternative-NHEJ (A-NHEJ) pathway. These findings suggest low expression of NME1 drives DSBR towards higher fidelity pathways, conferring enhanced genomic stability necessary for rapid and error-free proliferation in invasive and metastatic cells. The novel mechanism highlighted in the current study appears likely to impact metastatic potential and therapy-resistance in advanced melanoma and other cancers.
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