Molecular and Biochemical Characterization of <i>xrs</i> Mutants Defective in Ku80

Singleton, Belinda K.; Priestley, A.; Steingrimsdottir, Herdis; Gell, David A.; Blunt, Tracy; Jackson, Stephen D.; Lehmann, Alan R.; Jeggo, Penny A.

doi:10.1128/mcb.17.3.1264

Cited by 166 publications

(142 citation statements)

References 55 publications

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“…This agreed well with the results obtained by immunocytochemical analysis (Reeves, 1985;Yaneva et al, 1985;Mimori et al, 1986;Koike et al, 1998). Since both Ku70 and Ku80 monomers are highly unstable compared to their heterodimers (Errami et al, 1996;Gu et al, 1997;Singleton et al, 1997), we were interested in studying the nuclear translocation of Ku80 and its heterodimer partner Ku70. Osipovich et al (1997) reported that mutations in Ku80 corresponding to amino acids 453 and 454 abolished its ability to interact with Ku70, using an in vitro binding assay and the yeast two-hybrid analysis.…”

Section: Resultssupporting

confidence: 87%

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Ku80 can translocate to the nucleus independent of the translocation of Ku70 using its own nuclear localization signal

Koike¹,

Ikuta

Miyasaka³

et al. 1999

Oncogene

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Ku antigen is a complex of Ku70 and Ku80 subunits and plays an important role in not only DNA double-strand breaks (DSB) repair and V(D)J recombination, but also in growth regulation. Ku is generally believed to always form and function as heterodimers on the basis of in vitro observations. Here we demonstrate that the localization of Ku80 does not completely coincide with that of Ku70. Ku70 and Ku80 were colocalized in the nucleus in the interphase but not in the late telophase/early G1 phase of the cell cycle. Since the in vivo function of Ku might be partially regulated by the control of its transport, we attempted to investigate the molecular mechanisms underlying the nuclear translocation of Ku. The nuclear translocation of Ku80 started during the late telophase/ early G1 phase after the nuclear envelope was formed and this was preceded by the nuclear translocation of Ku70. Furthermore, we found that the Ku80 protein was transported to the nucleus without heterodimerization with Ku70. To understand in detail the mechanism of transport of Ku80, we attempted to identify the nuclear localization signal (NLS) of Ku80 and de®ned to a region spanning nine amino acid residues (positions 561 ± 569). The Ku80 NLS was demonstrated to be mediated to the nuclear rim by two components of PTAC58 and PTAC97. All these ®ndings support the idea that Ku80 can translocate to the nucleus using its own NLS independent of the translocation of Ku70.

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Section: Resultssupporting

confidence: 87%

“…Both Ku70 and Ku80 are highly unstable when expressed individually (Satoh et al, 1995). Loss of one of the Ku subunits results in a signi®cant decrease in the steady-state level of the other (Errami et al, 1996;Gu et al, 1997;Singleton et al, 1997). These ®ndings strongly suggest that Ku70 and Ku80 function as heterodimers.…”

Section: Introductionmentioning

confidence: 88%

Ku80 can translocate to the nucleus independent of the translocation of Ku70 using its own nuclear localization signal

Koike¹,

Ikuta

Miyasaka³

et al. 1999

Oncogene

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“…These data are in contrast to previous reports in rodents showing that the individual Ku subunits were degraded when not dimerized, because the Ku70 protein was undetectable in Ku86 rodent mutants. 50 In our study, the decrease in Ku DNA end-binding activity is potentially due to the reduction of the amount of Ku86 protein to substoechiometric levels with Ku70, because both subunits are equally necessary to form stable complexes in an EMSA. 28 The decrease in Ku DNA end-binding activity alone may contribute to the observed radiosensitivity and DSB repair defect.…”

Section: Discussionmentioning

confidence: 69%

Transfer of Ku86 RNA antisense decreases the radioresistance of human fibroblasts

et al. 2000

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Ku86 has been shown to be involved in DNA double-strand break (DSB) repair and radiosensitivity in rodents, but its role in human cells is still under investigation. The purpose of this study was to evaluate the radiosensitivity and DSB repair after transfection of a Ku86-antisense in a human fibroblast cell line. Simian virus 40-transformed MRC5V1 human fibroblasts were transfected with a vector (pcDNA3) containing a Ku86-antisense cDNA. The main endpoints were Ku86 protein level, Ku DNA end-binding and DNA protein kinase activity, clonogenic survival, and DSB repair kinetics. After transfection of the Ku86-antisense, decreased Ku86 protein expression, Ku DNA end-binding activity, and DNA protein kinase activity were observed in the uncloned cellular population. The fibroblasts transfected with the Ku86-antisense showed also a radiosensitive phenotype, with a surviving fraction at 2 Gy of 0.29 compared with 0.75 for the control and 20% of unrepaired DSB observed at 24 hours after irradiation compared with 0% for the control. Several clones were also isolated with a decreased level of Ku86 protein, a surviving fraction at 2 Gy between 0.05 and 0.40, and 10 -20% of unrepaired DSB at 24 hours. This study is the first to show the implication of Ku86 in DSB repair and in the radiosensitivity of human cells. This investigation strongly suggests that Ku86 could constitute an appealing target for combining gene therapy and radiation therapy. Cancer Gene Therapy (2000) 7, 339 -346

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“…In fact, it has been reported that Ku70-deficient mice exhibit decreased levels of Ku86, and vice versa. [25][26][27] We found that 72 h of transfection are sufficient to efficiently knockdown Ku70/86 ( Figure 3e). Quantitative real-time PCR and immunoblotting analyses showed that a significant increase of Apaf1 mRNA and protein levels occurred both in ImimPc-2 and in ETNA cells when compared with cells transfected for 72 h with an unrelated control oligonucleotide (Figure 3d and e).…”

Section: Resultsmentioning

confidence: 93%

The DNA repair complex Ku70/86 modulates Apaf1 expression upon DNA damage

et al. 2010

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Apaf1 is a key regulator of the mitochondrial intrinsic pathway of apoptosis, as it activates executioner caspases by forming the apoptotic machinery apoptosome. Its genetic regulation and its post-translational modification are crucial under the various conditions where apoptosis occurs. Here we describe Ku70/86, a mediator of non-homologous end-joining pathway of DNA repair, as a novel regulator of Apaf1 transcription. Through analysing different Apaf1 promoter mutants, we identified an element repressing the Apaf1 promoter. We demonstrated that Ku70/86 is a nuclear factor able to bind this repressing element and downregulating Apaf1 transcription. We also found that Ku70/86 interaction with Apaf1 promoter is dynamically modulated upon DNA damage. The effect of this binding is a downregulation of Apaf1 expression immediately following the damage to DNA; conversely, we observed Apaf1 upregulation and apoptosis activation when Ku70/86 unleashes the Apaf1-repressing element. Therefore, besides regulating DNA repair, our results suggest that Ku70/86 binds to the Apaf1 promoter and represses its activity. This may help to inhibit the apoptosome pathway of cell death and contribute to regulate cell survival.

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Molecular and Biochemical Characterization of xrs Mutants Defective in Ku80

Cited by 166 publications

References 55 publications

Ku80 can translocate to the nucleus independent of the translocation of Ku70 using its own nuclear localization signal

Ku80 can translocate to the nucleus independent of the translocation of Ku70 using its own nuclear localization signal

Transfer of Ku86 RNA antisense decreases the radioresistance of human fibroblasts

The DNA repair complex Ku70/86 modulates Apaf1 expression upon DNA damage

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