Accumulation of Ku80 proteins at DNA double-strand breaks in living cells

Koike, Manabu; Koike, Aki

doi:10.1016/j.yexcr.2007.11.014

Cited by 38 publications

(79 citation statements)

References 45 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Similar to the N terminus of BRCA1, Ku80 accumulated at DSBs rapidly after laser irradiation (Fig. 4B and 7B), consistent with previous reports (27,35,47) Ku70 and DNA-PKcs are only transiently localized to laserirradiated sites (25). In our analysis, the N terminus of BRCA1 also accumulated only transiently ( Fig.…”

Section: Discussionsupporting

confidence: 91%

Rapid Recruitment of BRCA1 to DNA Double-Strand Breaks Is Dependent on Its Association with Ku80

Wei

Lan

Zhang

et al. 2008

Molecular and Cellular Biology

View full text Add to dashboard Cite

BRCA1 is the first susceptibility gene to be linked to breast and ovarian cancers. Although mounting evidence has indicated that BRCA1 participates in DNA double-strand break (DSB) repair pathways, its precise mechanism is still unclear. Here, we analyzed the in situ response of BRCA1 at DSBs produced by laser microirradiation. The amino (N)-and carboxyl (C)-terminal fragments of BRCA1 accumulated independently at DSBs with distinct kinetics. The N-terminal BRCA1 fragment accumulated immediately after laser irradiation at DSBs and dissociated rapidly. In contrast, the C-terminal fragment of BRCA1 accumulated more slowly at DSBs but remained at the sites. Interestingly, rapid accumulation of the BRCA1 N terminus, but not the C terminus, at DSBs depended on Ku80, which functions in the nonhomologous end-joining (NHEJ) pathway, independently of BARD1, which binds to the N terminus of BRCA1. Two small regions in the N terminus of BRCA1 independently accumulated at DSBs and interacted with Ku80. Missense mutations found within the N terminus of BRCA1 in cancers significantly changed the kinetics of its accumulation at DSBs. A P142H mutant failed to associate with Ku80 and restore resistance to irradiation in BRCA1-deficient cells. These might provide a molecular basis of the involvement of BRCA1 in the NHEJ pathway of the DSB repair process.Germ line mutations in the breast cancer susceptibility gene BRCA1 predispose women to breast and ovarian cancers (26, 37). The human BRCA1 protein is composed of 1,863 amino acid (aa) residues and contains a RING domain in the N terminus. Two tandem BRCT domains, which are frequently found in DNA repair proteins and function as a binding module for phospho-serine peptides (34), are present in the C terminus. The N-terminal region of BRCA1 directly interacts with BARD1 (51), and the association with BARD1 enhances the ubiquitin polymerase activity of BRCA1 (2,11,33).BRCA1 is involved in many cellular processes, including DNA repair, transcription, cell cycle regulation, chromatin remodeling, and apoptosis. Several lines of evidences suggest that the involvement of BRCA1 in the DNA repair pathway is associated with the tumor suppressor activity of BRCA1. For example, mouse and human cells deficient in BRCA1 are more sensitive to DNA damage, including ionizing irradiation and drugs that produce double-strand breaks (DSBs) or interstrand cross-linking of DNA. Clinically observed missense mutations often result in a nonfunctional BRCA1 protein that has lost the ability to repair DNA damage (43). BRCA1 interacts with a number of DNA repair factors such as Rad51, the Mre11-Rad50-Nbs1 (MRN) complex, BLM, and the DNA helicase BACH1 (also called BRIP1or FANCJ) (8,42,50,57). BRCA1 localizes to nuclear foci during S phase of the cell cycle. Various types of DNA damage result in hyperphosphorylation of BRCA1 and alterations in BRCA1 localization to nuclear foci (21, 41). Normally, BRCA1 redistributes to nuclear foci where multiple DNA repair factors accumulate. BRCA1 colocalizes with phosphor...

show abstract

Section: Discussionsupporting

confidence: 91%

Rapid Recruitment of BRCA1 to DNA Double-Strand Breaks Is Dependent on Its Association with Ku80

Wei

Lan

Zhang

et al. 2008

Molecular and Cellular Biology

View full text Add to dashboard Cite

show abstract

“…The nuclear translocation of Ku70 is, at least in part, controlled at a nuclear localization signal (NLS)-recognition step by NLS receptors and regulated by the heterodimerization with Ku80 [12][13][14][15]17]. In hamster cells, Ku70/Ku80 heterodimer accumulation at DSBs produced using a 405-nm laser starts immediately after irradiation [18], strongly supporting the idea that the heterodimer of Ku70 and Ku80 is a sensor of DSBs in the nuclei. Most recently, we have reported that Ku80, in contrast to H2AX, is highly mobile in the nuclei of hamster cells and that the mobility of a major portion of Ku80 is not affected by DNA DSBs produced by treatment with antitumor drugs [19].…”

Section: Discussionmentioning

confidence: 78%

“…Furthermore, it was reported that Ku80 is detectable only in the kidneys of Ku70-/-mice, but at a reduced level compared with that of Ku70+/-mice, whereas Ku80 is undetectable in extracts from the brain, lungs and liver [5]. The heterodimerization of Ku70/Ku80 is essential for Ku80 accumulation at the DNA damage sites and Ku70-dependent DSB repair, i.e., the classical NHEJ pathway [8,18]. We speculate that Ku70 plays a key role in regulation of the Ku80 expression level in order to control the activity of Ku70-dependent NHEJ at the DSB recognition step.…”

Section: Discussionmentioning

confidence: 99%

“…The cells were grown in a monolayer culture in DMEM (Nissui Seiyaku Co., Tokyo, Japan) supplemented with 10% fetal bovine serum and were maintained in a humidified incubator at 37°C under 5% CO 2 . Transient transfections were performed in Ku70-/-cells using FuGene6 (Roche Diagnostics K.K., Indianapolis, IN, U.S.A.) as described previously, and the cells were cultured for 2 days and then monitored using an FV300 confocal laser scanning microscope (Olympus, Tokyo, Japan) as previously described [18,19].…”

Section: Mice Cell Lines Cultures and Transfectionmentioning

confidence: 99%

“…DNA was stained with DAPI fluorescent dye. The distribution of fluorescent signals was monitored using an FV300 confocal laser scanning microscope (Olympus) as previously described [18,19].…”

Section: Mice Cell Lines Cultures and Transfectionmentioning

confidence: 99%

See 2 more Smart Citations

Establishment of Ku70-Deficient Lung Epithelial Cell Lines and Their Hypersensitivity to Low-Dose X-Irradiation

Koike¹,

Yutoku

Koike

2011

The Journal of Veterinary Medical Science

Self Cite

View full text Add to dashboard Cite

ABSTRACT. In clinical situations, cellular resistance to chemotherapy and radiotherapy is a significant component of tumor treatment failure. The DNA repair protein Ku70 is a key contributor to chemoresistance to anticancer agents, e.g., etoposide and bleomycin, or radioresistance. Ku70 plays a key role as a sensor of DNA double-strand breaks (DSBs) induced following exposure to ionizing radiation as well as treatment with some chemotherapeutic drugs. The responses of different organs to radiation vary widely and likely depend on the cell population in the organs. However, it is not clear whether Ku70 plays a role in the low-dose radioresistance of lung epithelial cells. In this study, we established Ku70-deficient epithelial cell lines from murine lungs lacking Ku70. Ku70-/-lung epithelial cells exhibited reduced Ku80 expression. Moreover, Ku70-/-lung epithelial cells were more sensitive than controls (Ku70+/-lung epithelial cells) to low-dose X-irradiation (< 0.5 Gy). We also found that consistent with the Ku70 function as a sensor of DSBs, Ku70 mainly localized in the nuclei of murine lung epithelial cells. These findings clearly indicate that Ku70 plays a key role in regulation of the Ku80 expression level in and the radioresistance of lung epithelial cells. Our data also suggest that these cell lines might be useful not only for study of Ku70 functions and the DSB repair pathway, but also for study of the molecular mechanism underlying the sensitivity to chemotherapeutic drugs and radiation in lung epithelial cells.

show abstract

Cloning of canine Ku80 and its localization and accumulation at DNA damage sites

2017

Self Cite

View full text Add to dashboard Cite

Molecularly targeted therapies have high specificity and significant cancer‐killing effect. However, their antitumor effect might be greatly diminished by variation in even a single amino acid in the target site, as it occurs, for example, as a consequence of SNP s. Increasing evidence suggests that the DNA repair protein Ku80 is an attractive target molecule for the development of next‐generation radiosensitizers for human cancers. However, the localization, post‐translational modifications (PTMs), and complex formation of Ku80 have not been elucidated in canines. In this study, for the first time, we cloned, sequenced, and characterized canine Ku80 cDNA . Our data show that canine Ku80 localizes in the nuclei of interphase cells and is quickly recruited at laser‐induced double‐strand break sites. Comparative analysis shows that canine Ku80 had only 82.3% amino acid identity with the homologous human protein, while the nuclear localization signal ( NLS ) in human and canine Ku80 is evolutionarily conserved. Notably, some predicted PTM sites, including one acetylation site and one sumoylation site within the NLS , are conserved in the two species. These findings suggest that the spatial and temporal regulation of Ku80 might be conserved in humans and canines. However, our data indicate that the expression of Ku80 is considerably lower in the canine cell lines examined than in human cell lines. These important findings might be useful to better understand the mechanism of the Ku80‐dependent DNA repair and for the development of potential next‐generation radiosensitizers targeting common targets in human and canine cancers.

show abstract

Accumulation of Ku80 proteins at DNA double-strand breaks in living cells

Cited by 38 publications

References 45 publications

Rapid Recruitment of BRCA1 to DNA Double-Strand Breaks Is Dependent on Its Association with Ku80

Rapid Recruitment of BRCA1 to DNA Double-Strand Breaks Is Dependent on Its Association with Ku80

Establishment of Ku70-Deficient Lung Epithelial Cell Lines and Their Hypersensitivity to Low-Dose X-Irradiation

Cloning of canine Ku80 and its localization and accumulation at DNA damage sites

Contact Info

Product

Resources

About