Cell cycle-dependent migration of the DNA-binding protein Ku80 into nucleoli

Li, Lilan; Yeh, Ning-Hsing

doi:10.1016/0014-4827(92)90433-9

Cited by 25 publications

(13 citation statements)

References 21 publications

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“…DNA-PK complexes were isolated from HeLa cell nuclear extracts using a Ku80 immunoaffinity matrix as follows: monoclonal antibodies specific for Ku80 (34) were conjugated to protein G agarose beads using dimethylpimelimidate HCl to achieve a final concentration of 1 mg of antibody/ml of agarose beads. 50 ml of HeLa cell nuclear extract (10 mg/ml) was incubated with 2 ml of the anti-Ku80 matrix for 4 h at 4°C in the presence of 100 g/ml sheared salmon sperm DNA.…”

Section: Methodsmentioning

confidence: 99%

Stimulation of the DNA-dependent Protein Kinase by Poly(ADP-Ribose) Polymerase

Ruscetti¹,

Lehnert²,

Halbrook³

et al. 1998

Journal of Biological Chemistry

216

145

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The DNA-dependent protein kinase (DNA-PK) is a heterotrimeric enzyme that binds to double-stranded DNA and is required for the rejoining of double-stranded DNA breaks in mammalian cells. It has been proposed that DNA-PK functions in this DNA repair pathway by binding to the ends of broken DNA molecules and phosphorylating proteins that bind to the damaged DNA ends. Another enzyme that binds to DNA strand breaks and may also function in the cellular response to DNA damage is the poly(ADP-ribose) polymerase (PARP). Here, we show that PARP can be phosphorylated by purified DNA-PK, and the catalytic subunit of DNA-PK is ADP-ribosylated by PARP. The protein kinase activity of DNA-PK can be stimulated by PARP in the presence of NAD ؉ in a reaction that is blocked by the PARP inhibitor 1,5-dihydroxyisoquinoline. The stimulation of DNA-PK by PARP-mediated protein ADP-ribosylation occurs independent of the Ku70/80 complex. Taken together, these results show that PARP can modify the activity of DNA-PK in vitro and suggest that these enzymes may function coordinately in vivo in response to DNA damage.Biochemical pathways that function in the recognition and repair of DNA damage are critical for maintaining genomic integrity. Potentially devastating DNA damage in the form of double-strand breaks (DSBs) 1 can occur when cells are exposed to ionizing radiation, oxidative stress and radiomimetic drugs. The DNA-dependent protein kinase (DNA-PK) is a key component of DNA DSB rejoining pathways in mammalian cells. DNA-PK is a heterotrimeric enzyme complex comprised of a 460-kDa catalytic subunit (1) and a regulatory component consisting of the Ku70 and Ku80 proteins (2, 3). The Ku70 and Ku80 proteins form a heterodimeric complex that binds to the ends of double-stranded DNA with high affinity (4 -8). The catalytic subunit of DNA-PK (DNA-PKcs) binds to the Ku70/80 complex in the presence of double-stranded DNA (9) and phosphorylates a wide variety of protein substrates in vitro on serine and threonine residues (10, 11). The protein kinase activity of DNA-PK is autoregulatory; in the absence of a phosphorylation substrate, DNA-PKcs autophosphorylates and dissociates from the Ku70/80-DNA complex (12).Evidence that DNA-PK plays an integral role in the repair of DNA DSB has been provided through the characterization of rodent cell lines that have mutations that disrupt the expression of the Ku80 (13-19) or DNA-PKcs (20 -24). Although it is clear that DNA-PK is an important component of mammalian DNA DSB repair pathways, it is not known how the enzyme participates in these processes. In vitro, DNA-PK preferentially phosphorylates protein substrates that co-localize on the same DNA molecule (3,25). This suggests that the specificity of the phosphorylation reaction may be regulated, in part, via the co-localization of the enzyme and substrate target on DNA. Based on these data, it has been proposed that DNA-PK could participate in the DNA rejoining reaction by phosphorylating DNA repair factors that co-localize with it on broken DNA en...

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Section: Methodsmentioning

confidence: 99%

Stimulation of the DNA-dependent Protein Kinase by Poly(ADP-Ribose) Polymerase

Ruscetti¹,

Lehnert²,

Halbrook³

et al. 1998

Journal of Biological Chemistry

216

145

View full text Add to dashboard Cite

show abstract

“…Ku70͞86 was purified from HeLa cell nuclear extracts using an anti-Ku80 antibody affinity column. Antibodies specific for Ku86 (47) were conjugated to protein G agarose beads using dimethylpimelimidate HCl to achieve a final concentration of 1 mg antibody͞ml. HeLa cell nuclear extract (50 ml) (48) containing 500 mg of protein was incubated with 2 ml of the anti-Ku80 matrix for 4 hr at 4ЊC in the presence of 100 g͞ml sheared salmon sperm DNA.…”

Section: Methodsmentioning

confidence: 99%

DNA looping by Ku and the DNA-dependent protein kinase

Cary

Peterson

Wang

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

228

138

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The DNA-dependent protein kinase (DNA-PK) is required for DNA double-strand break (DSB) repair and immunoglobulin gene rearrangement and may play a role in the regulation of transcription. The DNA-PK holoenzyme is composed of three polypeptide subunits: the DNA binding Ku70͞86 heterodimer and an Ϸ460-kDa catalytic subunit (DNA-PKcs). DNA-PK has been hypothesized to assemble at DNA DSBs and play structural as well as signal transduction roles in DSB repair. Recent advances in atomic force microscopy (AFM) have resulted in a technology capable of producing high resolution images of native protein and proteinnucleic acid complexes without staining or metal coating. The AFM provides a rapid and direct means of probing the protein-nucleic acid interactions responsible for DNA repair and genetic regulation. Here we have employed AFM as well as electron microscopy to visualize Ku and DNA-PK in association with DNA. A significant number of DNA molecules formed loops in the presence of Ku. DNA looping appeared to be sequence-independent and unaffected by the presence of DNA-PKcs. Gel filtration of Ku in the absence and the presence of DNA indicates that Ku does not form nonspecific aggregates. We conclude that, when bound to DNA, Ku is capable of self-association. These findings suggest that Ku binding at DNA DSBs will result in Ku self-association and a physical tethering of the broken DNA strands.

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“…Ku antigen (Ku86/70) is another nucleolar protein that undergoes redistribution during mitosis (Li and Yeh, 1992). Here we observed that Ku86 remained associated with nucleolar regions at early prophase when chromosomes had already started to condense, which was in contrast to an apparent extrusion of TRF2 from the nucleolus (Fig.…”

Section: Trf2 Is Localized In the Nucleolus Of Human Cellsmentioning

confidence: 99%

Nucleolar localization of the human telomeric repeat binding factor 2 (TRF2)

Zhang

Hemmerich

Grosse

2004

Journal of Cell Science

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The telomeric repeat binding factor 2 (TRF2) specifically recognizes TTAGGG tandem repeats at chromosomal ends. Unexpectedly immunofluorescence studies revealed a prominent nucleolar localization of TRF2 in human cells, which appeared as discrete dots with sizes similar to those present in the nucleoplasm. The TRF2 dots did not overlap with dots stemming from the upstream binding factor (UBF) or the B23 protein. After treatment with a low concentration of actinomycin D (0.05 μg/ml), TRF2 remained in the nucleolus, although this condition selectively inhibited RNA polymerase I and led to a relocalization of UBF and B23. TRF2 was prominent in the nucleolus at G0 and S but seemed to diffuse out of the nucleolus in G2 phase. During mitosis TRF2 dispersed from the condensed chromosomes and returned to the nucleolus at cytokinesis. Treatment with low doses of actinomycin D delayed the release of TRF2 from the nucleolus as cells progressed from G2 phase into mitosis. With actinomycin D present TRF2 was detected in discrete foci adjacent to UBF in prophase, while in metaphase a complete overlap between TRF2 and UBF was observed. TRF2 was present in DNase-insensitive complexes of nucleolar extracts, whereas DNA degradation disrupted the protein-DNA complexes consisting of Ku antigen and B23. Following treatment with actinomycin D some of the mitotic cells displayed chromosome end-to-end fusions. This could be correlated to the actinomycin D-suppressed relocalization of TRF2 from the nucleolus to the telomeres during mitosis. These results support the view that the nucleolus may sequester TRF2 and thereby influences its telomeric functions.

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Cell cycle-dependent migration of the DNA-binding protein Ku80 into nucleoli

Cited by 25 publications

References 21 publications

Stimulation of the DNA-dependent Protein Kinase by Poly(ADP-Ribose) Polymerase

Stimulation of the DNA-dependent Protein Kinase by Poly(ADP-Ribose) Polymerase

DNA looping by Ku and the DNA-dependent protein kinase

Nucleolar localization of the human telomeric repeat binding factor 2 (TRF2)

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