Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Repair of DNA double-strand breaks by the non-homologous end-joining pathway (NHEJ) requires a minimal set of proteins including DNA-dependent protein kinase (DNA-PK), DNA-ligase IV and XRCC4 proteins. DNA-PK comprises Ku70/Ku80 heterodimer and the kinase subunit DNA-PKcs (p460). Here, by monitoring protein assembly from human nuclear cell extracts on DNA ends in vitro, we report that recruitment to DNA ends of the XRCC4-ligase IV complex responsible for the key ligation step is strictly dependent on the assembly of both the Ku and p460 components of DNA-PK to these ends. Based on co-immunoprecipitation experiments, we conclude that interactions of Ku and p460 with components of the XRCC4-ligase IV complex are mainly DNA-dependent. In addition, under p460 kinase permissive conditions, XRCC4 is detected at DNA ends in a phosphorylated form. This phosphorylation is DNA-PK-dependent. However, phosphorylation is dispensable for XRCC4-ligase IV loading to DNA ends since stable DNA-PK/XRCC4-ligase IV/DNA complexes are recovered in the presence of the kinase inhibitor wortmannin. These findings extend the current knowledge of the assembly of NHEJ repair proteins on DNA termini and substantiate the hypothesis of a scaffolding role of DNA-PK towards other components of the NHEJ DNA repair process.
J Mol Biol 2003 Feb 07
PMID:Coordinated assembly of Ku and p460 subunits of the DNA-dependent protein kinase on DNA ends is necessary for XRCC4-ligase IV recruitment. 1254 93

The Ku autoantigen/KARP-1 (Ku86 autoantigen related protein-1) plays an important role in the double-strand break repair of mammalian DNA as a DNA-binding component of DNA-dependent protein kinase (DNA-PK) complex. KARP-1 is differently transcribed from the human Ku86 autoantigen gene locus and it is implicated in the control of DNA-dependent protein kinase activity. We cloned rKAB1, a rat homolog of KAB1 (KARP-1 binding protein 1 of human) from a rat hippocampal cDNA library. rKAB1 mRNA was specifically expressed in the brain and the thymus. EGFP-tagged rKAB1 protein localized in cell nucleus and in the condensed chromosome during the mitotic cell division. We found that rKAB1 works as a protective protein against cell damage by oxidative stress.
Mol Cell Biochem 2003 Jun
PMID:Molecular cloning and characterization of rKAB1, which interacts with KARP-1, localizes in the nucleus and protects cells against oxidative death. 1287 Jun 57

The homologous recombination (HR) DNA repair pathway participates in telomere length maintenance in yeast but its putative role at mammalian telomeres is unknown. Mammalian Rad54 is part of the HR machinery, and Rad54-deficient mice show a reduced HR capability. Here, we show that Rad54-deficient mice also show significantly shorter telomeres than wild-type controls, indicating that Rad54 activity plays an essential role in telomere length maintenance in mammals. Rad54 deficiency also resulted in an increased frequency of end-to-end chromosome fusions involving telomeres compared to the controls, suggesting a putative role of Rad54 in telomere capping. Finally, the study of mice doubly deficient for Rad54 and DNA-PKcs showed that telomere fusions due to DNA-PKcs deficiency were not rescued in the absence of Rad54, suggesting that they are not mediated by Rad54 activity.
Mol Cell Biol 2003 Aug
PMID:Role of mammalian Rad54 in telomere length maintenance. 1289 31

The DNA-dependent protein kinase (DNA-PK) plays an essential role in nonhomologous DNA end joining (NHEJ) by initially recognizing and binding to DNA breaks. We have shown that in vitro, purified DNA-PK undergoes autophosphorylation, resulting in loss of activity and disassembly of the kinase complex. Thus, we have suggested that autophosphorylation of the DNA-PK catalytic subunit (DNA-PKcs) may be critical for subsequent steps in DNA repair. Recently, we defined seven autophosphorylation sites within DNA-PKcs. Six of these are tightly clustered within 38 residues of the 4,127-residue protein. Here, we show that while phosphorylation at any single site within the major cluster is not critical for DNA-PK's function in vivo, mutation of several sites abolishes the ability of DNA-PK to function in NHEJ. This is not due to general defects in DNA-PK activity, as studies of the mutant protein indicate that its kinase activity and ability to form a complex with DNA-bound Ku remain largely unchanged. However, analysis of rare coding joints and ends demonstrates that nucleolytic end processing is dramatically reduced in joints mediated by the mutant DNA-PKcs. We therefore suggest that autophosphorylation within the major cluster mediates a conformational change in the DNA-PK complex that is critical for DNA end processing. However, autophosphorylation at these sites may not be sufficient for kinase disassembly.
Mol Cell Biol 2003 Aug
PMID:Autophosphorylation of the catalytic subunit of the DNA-dependent protein kinase is required for efficient end processing during DNA double-strand break repair. 1289 53

Hyperoxia has been shown to cause DNA damage resulting in growth arrest of cells in p53-dependent, as well as p53-independent, pathways. Although H2O2 and other peroxides have been shown to induce ataxia telangiectasia-mutated (ATM)-dependent p53 phosphorylation in response to DNA damage, the signal transduction mechanisms in response to hyperoxia are currently unknown. Here we demonstrate that hyperoxia phosphorylates the Ser15 residue of p53 independently of ATM. Hyperoxia phosphorylated p53 (Ser15) in DNA-dependent protein kinase null (DNA-PK-/-) cells, indicating that it may not depend on DNA-PK for phosphorylation of p53 (Ser15). We show that Ser37 and Ser392 residues of p53 are also phosphorylated in an ATM-independent manner in hyperoxia. In contrast, H2O2 did not phosphorylate Ser37 in either ATM+/+ or ATM-/- cells. Furthermore, H2O2 failed to phosphorylate Ser15 in ATM-/- cells. Additionally, overexpression of kinase-inactive ATM-and-Rad3-related (ATR) in HEK293T cells diminished Ser15, Ser37, and Ser392 phosphorylation compared with vector-only transfected cells. In contrast, wild-type ATR overexpression did not diminish Ser15, Ser37, or Ser392 phosphorylation. We also show that checkpoint kinase 1 (Chk1) is phosphorylated on Ser345 in response to hyperoxia, which could be inhibited by caffeine or wortmannin, potent inhibitors of phosphoinositide 3-kinase-related kinases. Hyperoxia also phosphorylated Chk1 in ATM+/+ as well as in ATM-/- cells, demonstrating an ATM-independent mechanism in Chk1 phosphorylation. Together, our data suggest that hyperoxia activates the ATR-Chk1 pathway and phosphorylates p53 at multiple sites in an ATM-independent manner, which is different from other forms of oxidative stress such as H2O2 or UV light.
Am J Physiol Lung Cell Mol Physiol 2004 Jan
PMID:Hyperoxia activates the ATR-Chk1 pathway and phosphorylates p53 at multiple sites. 1295 29

DNA-dependent protein kinase (DNA-PK), composed of Ku70, Ku80, and the catalytic subunit (DNA-PKcs), is involved in double-strand break (DSB) repair by non-homologous end joining (NHEJ). DNA-PKcs defects confer ionizing radiation sensitivity and increase homologous recombination (HR). Increased HR is consistent with passive shunting of DSBs from NHEJ to HR. We therefore predicted that inhibiting the DNA-PKcs kinase would increase HR. A novel DNA-PKcs inhibitor (1-(2-hydroxy-4-morpholin-4-yl-phenyl)-ethanone; designated IC86621) increased ionizing radiation sensitivity but surprisingly decreased spontaneous and DSB-induced HR. Wortmannin also inhibits DNA-PKcs and reduces DSB-induced HR. IC86621 did not affect HR product outcome, indicating that it affects HR initiation. Thus, HR is increased in the absence of DNA-PKcs, but decreased when DNA-PKcs is catalytically inactive, suggesting interactive competition between HR and NHEJ. The effects of IC86621 and wortmannin were proportional to the level of DNA-PKcs, consistent with inhibited DNA-PKcs acting in a dominant negative manner. We propose that inhibition of DNA-PKcs blocks its autophosphorylation, prevents dissociation of DNA-PKcs from DNA ends, and thereby blocks both HR and NHEJ. By blocking the two major DSB repair pathways, DNA-PKcs inhibitors should radiosensitize at all cell-cycle stages and are therefore excellent candidates for augmenting cancer radiotherapy.
Mol Cancer Res 2003 Oct
PMID:Interactive competition between homologous recombination and non-homologous end joining. 1457 92

Herein, we report that the DNA-dependent protein kinase (DNA-PK) regulates the DNA damage introduced during Sleeping Beauty (SB) element excision and reinsertion in mammalian cells. Using both plasmid- and chromosome-based mobility assays, we analyzed the repair of transposase-induced double-stranded DNA breaks in cells deficient in either the DNA-binding subunit of DNA-PK (Ku) or its catalytic subunit (DNA-PKcs). We found that the free 3' overhangs left after SB element excision were efficiently and accurately processed by the major Ku-dependent nonhomologous-end-joining pathway. Rejoining of broken DNA molecules in the absence of Ku resulted in extensive end degradation at the donor site and greatly increased the frequency of recombination with ectopic templates. Therefore, the major DNA-PK-dependent DNA damage response predominates over more-error-prone repair pathways and thereby facilitates high-fidelity DNA repair during transposon mobilization in mammalian cells. Although transposable elements were not found to be efficiently circularized after transposase-mediated excision, DNA-PK deficiency supported more-frequent transposase-mediated element insertion than was found in wild-type controls. We conclude that, based on its ability to regulate excision site junctional diversity and transposon insertion frequency, DNA-PK serves an important protective role during transpositional recombination in mammals.
Mol Cell Biol 2003 Dec
PMID:Nonhomologous-end-joining factors regulate DNA repair fidelity during Sleeping Beauty element transposition in mammalian cells. 1461 96

Functional inactivation of the p53 gene and robust DNA repair capacity may be among the salient causes of radioresistance in tumor cells. We expressed the wild-type (wt) p53 gene in a p53-mutant human epidermoid carcinoma cell line, A431, using an adenoviral vector [adenovirus-p53 (Ad-p53), INGN 201], examined its radiosensitivity, and correlated p53 status and radiosensitivity with cellular repair functions. Using clonogenic survival assays and the terminal deoxynucleotidyl transferase-mediated nick end labeling assay for apoptosis, we demonstrated that preirradiation treatment with Ad-p53 significantly increased the radiosensitivity of A431 cells over controls. Induction of p53 expression using a construct where p53 expression was under the control of an inducible promoter also significantly increased radiosensitivity of H1299 lung tumor cells, which are otherwise null for p53. These results did not correlate with radiation-induced apoptosis but did correlate with functional impairment of DNA repair and suppressed expression of several repair-related genes, such as Ku70, DNA-dependent protein kinase, ataxia telangiectasia mutated, and X-ray-sensitive complementation group 4. Normal human fibroblast MRC-9 cells showed no impairment in the repair capability due to Ad-p53 despite the suppression of some repair genes. Expression of Ku70, which is known to mediate diverse cellular functions, correlated with the differential effects of p53 on radiosensitivity in the normal and tumor cells.
Mol Cancer Ther 2003 Nov
PMID:Adenovirus-mediated wild-type p53 radiosensitizes human tumor cells by suppressing DNA repair capacity. 1461 96

In eukaryotes the non-homologous end-joining repair of double strand breaks in DNA is executed by a series of proteins that bring about the synapsis, preparation and ligation of the broken DNA ends. The mechanism of this process appears to be initiated by the obligate heterodimer (Ku70/Ku86) protein complex Ku that has affinity for DNA ends. Ku then recruits the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). The three-dimensional structures of the major part of the Ku heterodimer, representing the DNA-binding core, both free and bound to DNA are known from X-ray crystallography. However, these structures lack a region of ca 190 residues from the C-terminal region (CTR) of the Ku86 subunit (also known as Lupus Ku autoantigen p86, Ku80, or XRCC5) that includes the extreme C-terminal tail that is reported to be sufficient for DNA-PKcs-binding. We have examined the structural characteristics of the Ku86CTR protein expressed in bacteria. By deletion mutagenesis and heteronuclear NMR spectroscopy we localised a globular domain consisting of residues 592-709. Constructs comprising additional residues either to the N-terminal side (residues 543-709), or the C-terminal side (residues 592-732), which includes the putative DNA-PKcs-binding motif, yielded NMR spectra consistent with these extra regions lacking ordered structure. The three-dimensional solution structure of the core globular domain of the C-terminal region of Ku86 (Ku86CTR(592-709)) has been determined using heteronuclear NMR spectroscopy and dynamical simulated annealing using structural restraints from nuclear Overhauser effect spectroscopy, and scalar and residual dipolar couplings. The polypeptide fold comprises six regions of alpha-helical secondary structure that has an overall superhelical topology remotely homologous to the MIF4G homology domain of the human nuclear cap binding protein 80 kDa subunit and the VHS domain of the Drosophila protein Hrs, though strict analysis of the structures suggests that these domains are not functionally related. Two prominent hydrophobic pockets in the gap between helices alpha2 and alpha4 suggest a potential ligand-binding characteristic for this globular domain.
J Mol Biol 2004 Jan 09
PMID:The 3D solution structure of the C-terminal region of Ku86 (Ku86CTR). 1467 64

Extracts of Xenopus eggs and of cultured human and hamster cells have the capacity to join nonhomologous DNA ends, and all do so with similar specificity. To examine the formation of repair complexes on DNA under conditions of end joining, end-labeled fragments were incubated with the various extracts and then subjected to DNase-I footprinting. Human and Xenopus extracts produced footprints virtually identical to that of purified DNA-dependent protein kinase holoenzyme (Ku plus DNA-PKcs), with protection of the terminal 28 bp. Extracts of hamster cells were more variable, but usually produced a 16-bp footprint, similar to that of Ku alone. In all cases a 28-bp holoenzyme-like footprint was associated with wortmannin-sensitive end joining, minimal 3'-5' exonucleolytic resection, and a predominance of accurate end-joining products. To determine whether the short segments of DNA occupied by Ku and DNA-PK were sufficient to support end joining, Y-shaped substrates were constructed in which only one arm was available for end joining. A Y substrate with a 31-bp arm bearing a partially cohesive 3' overhang was accurately joined by a Xenopus egg extract, whereas a substrate with a 21-bp arm was not. Surprisingly, a human cell extract did not join the Y substrates at all. The results suggest that differences in wortmannin sensitivity and in the distribution of in vitro end-joining products may be attributable to the variations in the levels of DNA-PKcs in the extracts. In addition, end joining in human extracts appears to involve interactions with significantly longer segments of DNA than the approximately 28 bp occupied by DNA-PK.
Environ Mol Mutagen 2003
PMID:DNA end sequestration by DNA-dependent protein kinase and end joining of sterically constrained substrates in whole-cell extracts. 1467 73


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