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PubMed1993

A protein homologous to human Ku p70-protein is required for reconstitution of Xenopus sperm pronuclei.

The monoclonal antibody mAbH6, which recognizes one human Ku-protein (p70), cross-reacted with the counterpart protein from Xenopus eggs. The Xenopus antigen purified with a mAbH6-Sepharose column was a complex of 88 kDa and 72 kDa proteins. The role of Ku protein in nuclear structure formation was studied using a cell-free nuclear assembly extract derived from Xenopus interphase eggs. The protein was distributed on the surface of demembranated sperm chromatin and in the membrane vesicle fraction of the nuclear assembly extract. Addition of mAbH6 to the assembly extracts prevented the completion of reconstitution of pronuclei from demembranated sperm chromatins, although partial decondensation mediated by nucleoplasmin was not impeded. As a result, the sperm chromatin remained in partially swollen structures or formed round but small anomalous nuclei. Nuclear membranes were formed on the nuclei in mAbH6-inhibited extract systems, but DNA synthesis was largely decreased, suggesting the incomplete reconstitution of pronuclei. The incorporation of lamin to the nuclei was inhibited by mAbH6. It is suggested that the Xenopus Ku-homologous protein has a role in the formation of the lamin layer after nuclear membrane reconstitution.

Amino Acid Sequence↗
PubMed1995

Characterization of the HeLa cell single-stranded DNA-dependent ATPase/DNA helicase II.

A single-stranded DNA-dependent ATPase activity, consisting of two subunits of 83 kDa (p90) and 68 kDa (p70), was previously purified from HeLa cells (Vishwanatha, J.K. and Baril, E.F. (1990) Biochem 29, 8753-8759). Homology of the two subunits of single-stranded DNA-dependent ATPase with the human Ku protein (Cao et al. (1994) Biochem 33, 8548-8557) and identity of the Ku protein as the human DNA helicase II (Tuteja et al. (1994) EMBO J. 13, 4991-5001) have been reported recently. Using antisera raised against the subunits of the HDH II, we confirm that the Hela single-stranded DNA-dependent ATPase is the HDH II. Similar to the activity reported for Ku protein, ssDNA-dependent ATPase binds to double-stranded DNA and the DNA-protein complex detected by gel mobility shift assay consists of both the ATPase subunits. The p90 subunit is predominantly nuclear and is easily dissociated from chromatin. The p70 is distributed in cytosol and nucleus, and a fraction of the nuclear p70 protein is found to be associated with the nuclear matrix. Both the p90 and p70 subunits of the ATPase are present in G1 and S phase of the cell cycle and are rapidly degraded in the G2/M phase of the cell cycle.

Adenosine Triphosphatases↗
PubMed1990

The presence of an antigen reactive with a human autoantibody in Trichosia pubescens (Diptera: Sciaridae) and its association with certain transcriptionally active regions of the genome.

The antigens in HeLa and Trichosia pubescens cells, recognized by sera from patients with rheumatic diseases containing anti-Ku antibodies, were compared by means of immunoprecipitation of labeled cell extracts. The autoantibodies present in the tested sera precipitate at least two polypeptides of approximately Mr = 70,000 and Mr = 80,000 in HeLa cell extracts and a polypeptide of approximately Mr = 72,000 in Trichosia salivary gland cell extracts. The distribution of the insect antigen in chromatin was studied in salivary gland polytene chromosomes by indirect immunofluorescent staining with sera from two different patients. Both sera react with certain transcriptionally active chromosomal sites. The presence of the antigen in polytene chromosomes is strictly dependent on transcription, as no reaction is observed in the same sites before or after gene activation. Other sites, such as the nucleolar organizing region, are very active in transcription but never reacted with the anti-Ku positive sera. These results show that the insect antigen is associated with transcription-related processes of a subset of the chromosomal loci of T. pubescens. The anti-Ku positive sera react with a highly conserved antigen, which may serve a very important and similar role in the cellular metabolism of both insect and mammalian cells.

Animals↗
PubMed1996

Ku-related antigens are associated with transcriptionally active loci in Chironomus polytene chromosomes.

Antigens of Chironomus reactive with human sera containing anti-Ku antibodies and also with specific antibodies to each Ku subunit were characterized by immunoblot analysis. Three main antigen species were identified in nuclear-enriched extracts from salivary gland cells of Chironomus thummi, ranging in Mr from 55000 to 67000. The nuclear localization of Ku-related antigens in the dipteran Chironomus was studied by immunofluorescent labeling in polytene chromosomes of the salivary glands. Balbiani rings, loci highly active in transcription, were found to be strongly labeled by anti-Ku antibodies. Sugar-induced changes in the activity of the Balbiani ring genes were accompanied by the redistribution of Ku-related antigens as visualized by their absence in regressed Balbiani ring loci, and continued presence only in those that were transcriptionally active. A drastic change in the distribution of Ku-related antigens was also observed when C. thummi larvae underwent heat treatment as the immunofluorescent staining was restricted to previously described heat shock puffs. Anti-Ku sera reacted in addition with several chromosomal bands in which the presence of RNA polymerase II was also immunologically detected. The results show that Chironomus antigens reactive with anti-Ku antibodies are related to transcription in polytene chromosomes.

Alcohols↗
PubMed1997

Rare microsatellite polymorphisms in the DNA repair genes XRCC1, XRCC3 and XRCC5 associated with cancer in patients of varying radiosensitivity.

DNA repair defects might contribute both to cancer progression and to the extreme reactions to radiotherapy observed in approximately 5% of patients. Polymorphic microsatellites in three DNA repair genes, XRCC1, XRCC3 and XRCC5, were analyzed for possible linkage to cancer status or clinical radiosensitivity. XRCC1, 3 and 5 proteins are involved in single-strand DNA break rejoining, recombinational repair, and double-strand DNA break rejoining respectively. Mendelianly inherited microsatellite polymorphisms in these genes were analyzed in three groups: volunteers with no cancer history; radiosensitive cancer patients; cancer patients with acceptable reactions to radiotherapy. Rare heterozygous alterations in all three gene regions were found solely in the cancer subpopulation. Association testing between these rare polymorphisms and cancer status revealed a significant association for XRCC1 (P = 0.005), and XRCC3 (P = 0.004). There was also an association between these polymorphisms and clinical radiosensitivity for XRCC1 (P = 0.03), and XRCC3 (P = 0.005).

Adult↗
PubMed2006

Expression profiling identifies novel candidate genes for ethanol sensitivity QTLs.

The Inbred Long Sleep (ILS) and Inbred Short Sleep (ISS) mouse strains have a 16-fold difference in duration of loss of the righting response (LORR) following administration of a sedative dose of ethanol. Four quantitative trait loci (QTLs) have been mapped in these strains for this trait. Underlying each of these QTLs must be one or more genetic differences (polymorphisms in either gene coding or regulatory regions) influencing ethanol sensitivity. Because prior studies have tended to focus on differences in coding regions, genome-wide expression profiling in cerebellum was used here to identify candidate genes for regulatory region differences in these two strains. Fifteen differentially expressed genes were found that map to the QTL regions and polymorphisms were identified in the promoter regions of four of these genes by direct sequencing of ILS and ISS genomic DNA. Polymorphisms in the promoters of three of these genes, Slc22a4, Rassf2, and Tax1bp3, disrupt putative transcription factor binding sites. Slc22a4 and another candidate, Xrcc5, have human orthologs that map to genomic regions associated with human ethanol sensitivity in genetic linkage studies. These genes represent novel candidates for the LORR phenotype and provide new targets for future studies into the neuronal processes underlying ethanol sensitivity.

Animals↗
PubMed2004

Down-regulation of Ku 70 and Ku 80 mRNA expression in transitional cell carcinomas of the urinary bladder related to tumor progression.

DNA-dependent protein kinase (DNA-PK) containing the regulatory subunits Ku 70 and Ku 80 plays a prominent role in the repair of double-stranded DNA breaks by a nonhomologous end-joining pathway maintaining genomic stability. In an attempt to elucidate the significance of the DNA-PK complex for human urothelial carcinogenesis, the expression of Ku 70 and Ku 80 was studied in 71 transitional cell carcinomas (TCC) of the urinary bladder of various grades and stages, and in relation to lifestyle and occupational bladder cancer risk factors. To analyse the mRNA expression of Ku 70 and Ku 80, real-time quantitative reverse transcription-polymerase chain reaction was used and the protein expression assessed by immunohistochemistry. Advanced high-grade, high-stage TCC expressed the mRNA of Ku 70 and Ku 80 at a lower level than superficial low-grade, low-stage carcinomas, suggesting down-regulation of the Ku system to be associated with progression of bladder cancer from a low to a high malignant potential. The protein expression of Ku 70 and Ku 80 was closely related and decreased consistently with increasing grades and stages, paralleling the expression of the mRNA. Among hazardous environmental bladder cancer risk factors, heavy consumption of coffee was associated with a twofold decreased Ku 70 and Ku 80 mRNA expression, whereas tobacco smoke did not substantially affect the activity of the Ku system, except for a trend towards a dose-response relationship in the expression of Ku 70 mRNA. There is some evidence that exposure to polycyclic hydrocarbons, paints and lacquer, and stone dust may modify the expression of Ku 70 mRNA. Although the underlying molecular genetic pathways are not yet clearly understood, our data indicate that down-regulation of the Ku system promotes progression of urothelial carcinogenesis to a more malignant and aggressive clinical behavior, presumably as a result of an impaired capacity for DNA repair.

Antigens, Nuclear↗
PubMed2005

Telomere biology: integrating chromosomal end protection with DNA damage response.

Telomeres play the key protective role at chromosomes. Many studies indicate that loss of telomere function causes activation of DNA damage response. Here, we review evidence supporting interdependence between telomere maintenance and DNA damage response and present a model in which these two pathways are combined into a single mechanism for protecting chromosomal integrity. Proteins directly involved in telomere maintenance and DNA damage response include Ku, DNA-PKcs, RAD51D, PARP-2, WRN and RAD50/MRE11/NBS1 complex. Since most of these proteins participate in the repair of DNA double-strand breaks (DSBs), this was perceived by many authors as a paradox, given that telomeres function to conceal natural DNA ends from mechanisms that detect and repair DSBs. However, we argue here that the key function of one particular DSB protein, Ku, is to prevent or control access of telomerase, the enzyme that synthesises telomeric sequences, to both internal DSBs and natural chromosomal ends. This view is supported by observations that Ku has a high affinity for DNA ends; it acts as a negative regulator of telomerase and that telomerase itself can target internal DSBs. Ku then directs other DSB repair/telomere maintenance proteins to either repair DSBs at internal chromosomal sites or prevent uncontrolled elongation of telomeres by telomerase. This model eliminates the above paradox and provides a testable scenario in which the role of DSB repair proteins is to protect chromosomal integrity by balancing repair activities and telomere maintenance. In our model, a close association between telomeres and different DNA damage response factors is not an unexpected event, but rather a logical result of chromosomal integrity maintenance activities.

Animals↗
PubMed1998

Telomerase, Ku, and telomeric silencing in Saccharomyces cerevisiae.

Telomeres comprise a specialized chromosome end structure distinct from the standard nucleosomal architecture of the remainder of the genome. Telomere maintenance and chromosome stability require both replication of telomeric sequences by telomerase and telomeric end protection through binding of proteins. We have shown that Cdc13p and the heterodimer Ku are required, along with telomerase, for full telomere function, and we have proposed that Ku and Cdc13p contribute distinct roles in end protection. Ku has recently been shown to exhibit defects in transcriptional repression of telomere-proximal genes, known as telomere position effect (TPE), or telomeric silencing. We investigate here whether alterations in genes involved in the telomerase pathway also exhibit TPE defects and find that deletion or overexpression of EST1 or EST2 does not significantly affect telomeric silencing. However, telomeric silencing is derepressed upon overexpression of certain nonfunctional alleles of each. In addition, we determined that overproduction of telomerase pathway components partially alleviates the TPE defect in hdf1Delta cells. This indicates that there is genetic crosstalk between these two telomere maintenance pathways, and suggests that overproduction of telomerase pathway components may at least partially compensate for the loss of Ku in maintaining telomeric silencing.

Antigens, Nuclear↗
PubMed2006

Enhanced gene targeting frequency in ku70 and ku80 disruption mutants of Aspergillus sojae and Aspergillus oryzae.

In the koji molds Aspergillus sojae and Aspergillus oryzae, exogenous DNA is integrated in the genome, in most cases irrespective of the sequence homology, suggesting that DNA integration occurs predominantly through a nonhomologous end joining pathway where two ku genes, namely, ku70 and ku80, play a key role. To determine the effect of ku gene disruption on the gene targeting frequency, we constructed ku70-, ku80-, and ku70-ku80-disrupted strains of A. sojae and A. oryzae. The gene targeting frequency of the tannase gene in ku70 and ku80 strains of both Aspergillus species was markedly enhanced as compared with that of the parental strains. The gene targeting frequency of the aflR and ku80 genes was also enhanced in an A. sojae ku70 background. Therefore, the koji mold strains with ku-disrupted genes will be excellent tools as hosts for efficient gene targeting.

Antigens, Nuclear↗
PubMed2000

Effects of HDF1 (Ku70) and HDF2 (Ku80) on spontaneous and DNA damage-induced intrachromosomal recombination in Saccharomyces cerevisiae.

The Ku heterodimer binds to the ends of double-stranded breaks (DSBs) in DNA, and is involved in nonhomologous end joining. HDF1 and HDF2, which have been identified in Saccharomyces cerevisiae as homologues of the Ku70 and Ku80 proteins of mammals, reduce radiosensitivity only when homologous recombination repair is impaired and, therefore, affect DSB repair via nonhomologous recombination. Although it has been reported that homologous recombination is defective in the hdf1 null mutant, the roles of HDF1 and HDF2 in this process are not completely clear. We investigated the effect of HDF1 and HDF2 on intrachromosomal recombination by measuring rates of deletion between direct repeats caused by spontaneous and DNA damage-induced events (DEL recombination). We found a decrease in spontaneous DEL recombination in both TCY5 (hdf1delta) and TCY6 (hdf2delta) strains, suggesting that HDF1 and HDF2 play a role in homologous recombination. As DEL recombination events may occur by sister chromatid conversion and/or single-strand annealing, which is initiated by DSBs, HDF1 and HDF2 may be required to recruit proteins to the damaged ends so as to promote single-strand annealing. The strains TCY5 and TCY6 are also defective in methylmethane sulfonate (MMS)- and X-ray-induced, but not in UV-induced DEL recombination. This confirms that HDF1 and HDF2 are required for the completion of DEL recombination by single strand annealing.

Antigens, Nuclear↗
PubMed2006

DNA repair Ku proteins in gastric cancer cells and pancreatic acinar cells.

The DNA repair protein Ku acts as a heterodimer of Ku70 and Ku80 that binds to the DNA ends, nicks, or single-to-double-strand transition. It has a crucial role for DNA double-strand-break repair. Either Ku70 or Ku80 itself may have a unique function that is independent of the other Ku subunit. In this review, the role of Ku on cell proliferation and apoptosis will be discussed. Ku acts as a regulator of transcription by interacting with the recombination signal binding protein Jkappa and the NF-kappaB p50 homodimer to up-regulate p50 expression, which may regulate the proliferation of gastric cancer cells. Both Ku70 and Ku80 expressions are mediated by constitutively activated NF-kappaB and constitutively expressed cyclooxygenase-2 in gastric cancer cells, which may be related to gastric cell proliferation and carcinogenesis. In addition, nuclear loss of Ku may underlie the mechanism of apoptosis in pancreatic acinar cells after oxidative stress.

Animals↗
PubMed2007

Functional and genetic analysis of the Saccharomyces cerevisiae RNC1/TRM2: evidences for its involvement in DNA double-strand break repair.

We previously isolated the RNC1/TRM2 gene and provided evidence that it encodes a protein with a possible role in DNA double strand break repair. RNC1 was independently re-isolated as the TRM2 gene encoding a methyl transferase involved in tRNA maturation. Here we show that Trm2p purified as a fusion protein displayed 5' --> 3' exonuclease activity on double-strand (ds) DNA, and endonuclease activity on single-strand (ss) DNA, properties characteristic of previously isolated endo-exonucleases. A variant of Trm2p, Trm2p(ctDelta76aa) lacking 76 amino acids at the C-terminus retained nuclease activities but not the methyl transferase activity. Both the native and the variant exhibited sensitivity to the endo-exonuclease inhibitor pentamidine. The Saccharomyces cerevisiae trm2(Delta232-1920nt) mutant (containing only the first 231 nucleotides of the TRM2 gene) displayed low sensitivity to methyl methane sulfonate (MMS) and suppressed the MMS sensitivity of rad52 mutants in trm2(Delta232-1920nt)rad52 double mutants. The deletion of KU80, in trm2(Delta232-1920nt) mutant background displayed higher MMS sensitivity supporting the view of the possible role of Trm2p in a competing repair pathway separate from NHEJ. In addition, trm2 exo1 double mutants were synergistically more sensitive to MMS and ionizing radiation than either of the single mutant suggesting that TRM2 and EXO1 can functionally complement each other. However, the C-terminal portion, required for its methyl transferase activity was found not important for DNA repair. These results propose an important role for TRM2 in DNA repair with a potential involvement of its nuclease function in homologous recombination based repair of DNA DSBs.

Antigens, Nuclear↗
PubMed1992

Immunolocalization of Ku-proteins (p80/p70): localization of p70 to nucleoli and periphery of both interphase nuclei and metaphase chromosomes.

Distribution on both nuclei and metaphase chromosomes of Ku-proteins, recognized by autoantibodies from a patient with systemic lupus erythematosus, has been studied using a specific monoclonal antibody (mAbH6) that recognizes p70, one Ku-protein. Observation with either a conventional fluorescent microscope or a confocal laser scanning microscope revealed mAbH6-stained p70 antigen localized on both nuclear periphery and nucleoli of human interphase cells. The specific staining of nucleoli with mAbH6 has been confirmed using isolated nucleoli from rat liver in which the staining was seen as fine granules surrounding nucleolar DNA. During mitosis p70 antigen moved away from association with the nuclear envelope region to localization on the periphery of condensed chromosomes with no apparent staining of chromosome interior. The p70 antigen was copurified with DNA fragments by immunoaffinity column chromatography using mAbH6. The mAbH6 staining of both nuclear periphery and nucleoli was lost upon digestion with DNase I at low concentrations. These results suggest that p70 antigen is connected with these nuclear structures through DNA.

Amino Acid Sequence↗
PubMed1992

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

The DNA-binding protein Ku (p70/p80) was originally discovered through the use of human autoimmune sera. In attempts to search out nucleolar proteins in relation to nucleolar dynamic changes, we developed monoclonal antibodies against nuclear proteins. One antibody, termed LL1, received particular attention since asynchronous cells exhibited tremendous differences in their nucleolar fluorescence intensities after immunostaining. The LL1 protein was proven to be the Ku subunit p80 (Ku80) by cDNA cloning and sequencing. Possible correlations between the heterogeneous distribution of Ku80 in nucleoli and the cell cycle were examined. HeLa cells were synchronized at M phase by arrest with nocodazole, or at the G1/S boundary by sequential treatments with thymidine and aphidicolin. These cells were then released by culturing in fresh medium to allow the cell cycle to progress synchronously. Immunofluorescent detection of Ku80 revealed that nucleoli of the cells at the G1/S boundary had very small amounts of Ku80, which was mainly present in the nucleoplasm. Ku80 was gradually accumulated in nucleoli during S phase and reached the maximum at late S or G2 phase. Immunoblotting experiments showed that cell extracts prepared from different phases of the cell cycle had virtually identical amounts of Ku80. These results suggest that Ku80 migrates from nucleoplasm to nucleoli in a cell cycle-dependent manner.

Antibodies, Monoclonal↗
PubMed1991

Autoimmune antigen Ku is enriched on oligonucleotide columns distinct from those containing the octamer binding protein DNA consensus sequence.

During purification of the AP1 complex from the T cell line MLA144 we enriched for a complex which bound to an oligonucleotide column containing the AP1 DNA consensus sequence and co-eluted with a fraction required for AP1 binding activity. This complex although co-eluting with AP1 binding activity had previously been determined to be non-specific in its DNA binding properties. Further investigation determined that the complex was a heterodimer of 85 and 70 kDa which was antigenically related to the autoimmune antigen Ku. It is important to be aware of the abundance and avidity of the Ku complex to bind oligonucleotide columns when purifying sequence specific binding proteins.

Animals↗
PubMed1995

Altered metaphase chromosome structure in xrs-5 cells is not related to its radiation sensitivity or defective DNA break rejoining.

The chinese hamster ovary (CHO) cell line xrs-5 is a radiation-sensitive derivative of CHO-K1 cells. The xrs-5 cells have a defect in DNA double-strand break rejoining and show alterations in chromosome structure and nuclear morphology. The relationship between radiation sensitivity and metaphase chromosome morphology was examined in 12 'revertant' xrs-5 clones isolated following treatment with 5-azacytidine. nine of the clones were radioresistant while the other three retained xrs-5-like radiation sensitivity. Chromosome morphology reverted to CHO-K1-like characteristics in three of the radioresistant clones and one of the radiosensitive clones suggesting that the over-condensed metaphase chromosome morphology of xrs-5 cells does not underlie its radiation sensitivity. Radiation sensitivity did correlate with DNA double-strand break rejoining ability. The radioresistant clones showing the over-condensed xrs-5-like chromosome morphology were also slightly more sensitive to the topoisomerase II inhibitor etoposide (VP-16) than CHO-K1, suggesting that the over-condensed morphology might be due to alterations in the phosphorylation of chromatin proteins.

Animals↗
PubMed1993

The DNA-dependent protein kinase: requirement for DNA ends and association with Ku antigen.

The DNA-dependent protein kinase (DNA-PK) phosphorylates Sp1 and several other nuclear proteins. Here, we show that Sp1 and the DNA-PK must be colocalized on the same DNA molecule for efficient phosphorylation to occur. Interestingly, we find that the DNA-PK binds to and is activated by the ends of DNA molecules. Furthermore, we show that the DNA binding properties of the DNA-PK are identical to those of Ku, a well-characterized human autoimmune antigen. We demonstrate that the DNA-PK can be fractionated into two components, one of which is Ku and the other of which is a polypeptide of approximately 350 kd. DNA cross-linking and coimmunoprecipitation studies indicate that the catalytic 350 kd DNA-PK component is directed to DNA by protein-protein interactions with Ku. The implications of the unusual DNA binding mode and multicomponent nature of the DNA-PK are discussed.

Antigens, Nuclear↗