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PubMed1999

Dna-end binding activity of Ku in synchronized cells.

Three different types of cells were synchronized by various methods and DNA-end binding (DEB) activities of Ku were compared with asynchronous controls. In CHO K1 cells synchronized in G1 phase by serum starvation and in S phase by serum refeeding, DEB activity was reduced in S cells but remained unchanged in G1 cells. However, the same type of cells synchronized in G1/S phase by double thymidine block and in S phase by releasing the blockage, have the same DEB activity as asynchronous controls. A similar result was found in RKO and HeLa cells synchronized by the latter method. Arresting cells in mitosis with nocodazole also generated different cell cycle effects. Ku activity was reduced in CHO K1 and RKO cells, but not in HeLa cells after treatment with nocodazole. In phase-enriched cells separated by centrifugal elutriation, DEB activities were similar at different stages of the cell cycle in all three types of cells. Thus, different synchronization procedures can give very different values of Ku activity in a cell type-dependent manner. Results from elutriated cells are consistent, and suggest DEB activity of Ku does not change with the cell cycle.

Animals↗
PubMed1997

Cell cycle checkpoints and DNA repair in Nijmegen breakage syndrome.

Nijmegen breakage syndrome is characterized by a variable T cell and B cell immunodeficiency, growth failure, and an increased risk of malignancy. It is inherited in an autosomal recessive manner and is biochemically related to ataxia-telangiectasia. Cells from a patient with Nijmegen breakage syndrome were unable to arrest cell cycle progression after exposure to ionizing radiation, and BrdU incorporation into newly synthesized DNA was uninhibited, demonstrating that these cells have an aberrant response to radiation exposure. Although gross chromosomal breakage was observed, dinucleotide repeat segments were stable over time, suggesting that other types of DNA stability were not affected. DNA-PK activity, which is mediated by a protein related to the ataxia-telangiectasia gene product and is intimately involved in DNA repair and VDJ recombination, was normal in cells from an NBS patient. Therefore, cells from patients with Nijmegen breakage syndrome have an abnormal response to radiation exposure similar to that seen in ataxia-telangiectasia.

Abnormalities, Multiple↗
PubMed1997

Regulation of ribosomal RNA gene transcription during retinoic acid-induced differentiation of mouse teratocarcinoma cells.

We have examined the mechanism of regulation of rRNA synthesis in mouse F9 teratocarcinoma cells that were induced to differentiate by retinoic acid and dibutyryl cAMP. Ribosomal RNA (rRNA) synthesis was significantly reduced during differentiation of F9 cells into parietal endoderm cells. Nuclear run-on assay revealed that the rRNA gene transcription rates were reduced in differentiated cells, and this phenomenon could be mimicked by in vitro transcription assay using nuclear extracts prepared from F9 stem and F9 parietal endoderm cells. Analysis of the DNA-binding activities of two RNA polymerase I (pol I) transcription factors E1BF/Ku and UBF revealed decreased affinity for their cognate recognition sequences. Immunoblot analysis showed a marked reduction in the amounts of E1BF/Ku and UBF in the differentiated cells. Analysis of the steady-state RNA levels for the smaller subunit of E1BF/Ku and for UBF in differentiating F9 cells revealed decreased mRNA synthesis and increase in message level for the differentiation-specific marker laminin B1 with progression of the differentiated status of the cells. This study has demonstrated that differentiation of mouse F9 teratocarcinoma cells into parietal endoderm cells leads to diminished rRNA synthesis, which may be mediated by reduced DNA-binding activities and amounts of at least two pol I transcription factors.

Animals↗
PubMed1999

The nuclear localization signal of the human Ku70 is a variant bipartite type recognized by the two components of nuclear pore-targeting complex.

Ku protein is a complex of two subunits, Ku70 and Ku80. Ku is suspected to participate in both DNA double-strand break repair and transcription. Since both of these processes take place in the cell nucleus, we have been investigating the subcellular localization and nuclear transport of Ku proteins. In the present study, we analyzed the subcellular localization and nuclear localization signal (NLS) of Ku70. Fusion proteins of Ku70 and green fluorescent protein (GFP) transiently expressed in cells were clearly localized in the nuclei of interphase cells. Ku70 staining was distributed throughout both the nucleus and the cytoplasm in late telophase to early G1 phase cells. The NLS of Ku70 was located at the region composed of 18 amino acid residues (positions 539 to 556). This region overlapped with the Ku80-independent DNA-binding domain reported previously. The Ku70 NLS consisted of two basic subregions and a nonbasic intervening region. All the subregions were necessary for complete NLS activity. The amino acids in the nonbasic intervening region of Ku70 might be important for full NLS activity not only to provide sufficient length between the two separated clusters of basic amino acids but also to have an adequate amino acid sequence. All of the basic amino acid residues in the basic subregions were conserved among mammalian and avian homologues, confirming their importance in the nuclear translocation of Ku70. The structure of the Ku70 NLS resembled the consensus of a bipartite-type NLS. The Ku70 NLS was mediated to target to the nuclear rim by two components of the nuclear pore-targeting complex, PTAC58 and PTAC97.

Amino Acid Sequence↗
PubMed2002

Identification of telomere-dependent "senescence-like" arrest in mouse embryonic fibroblasts.

In contrast to human primary cells, mouse embryonic fibroblasts (MEF) do not show telomere shortening-mediated replicative senescence due to the fact that they have telomerase activity and show sufficiently long telomeres. Instead, it is now generally accepted that the "senescence-like" arrest that occurs in MEF after 5-10 divisions in culture is mediated by telomere-length-independent mechanisms generally referred to as stress. Using telomerase-deficient MEF Terc(-/-), we show here that telomere shortening to a critical length leads to a premature senescence-like arrest in MEF, as well as has a negative effect on spontaneous immortalization. Similarly, elimination of the telomere end-capping protein Ku86 also leads to a premature senescence-like arrest and has a negative effect on spontaneous immortalization. Both Terc(-/-) MEF with short telomeres and Ku86(-/-) MEF show dysfunctional telomeres, as indicated by similarly increased frequencies of end-to-end fusions. These results suggest that loss of telomere function is a general mechanism leading to cell arrest. These observations also indicate that telomere dysfunction is interfering with successful cell division and thus interferes with tumor formation. In summary, we have identified here two different ways to induce a telomere-dependent senescence-like arrest in MEF.

Animals↗
PubMed1995

A YAC contig encompassing the XRCC5 (Ku80) DNA repair gene and complementation of defective cells by YAC protoplast fusion.

The Chinese hamster ovary xrs mutants are sensitive to ionizing radiation, defective in DNA double-strand break rejoining, and unable to carry out V(D)J recombination effectively. Recently, the gene defective in these mutants, XRCC5, has been shown to encode Ku80, a component of the Ku protein and DNA-dependent protein kinase. We present here a YAC contig involving 25 YACs mapping to the region 2q33-q34, which encompasses the XRCC5 gene. Eight new markers for this region of chromosome 2 are identified. YACs encoding the Ku80 gene were transferred to xrs cells by protoplast fusion, and complementation of all the defective phenotypes has been obtained with two YACs. We discuss the advantages and disadvantages of this approach as a strategy for cloning human genes complementing defective rodent cell lines.

Animals↗
PubMed1996

Chromosomal localization of the mouse and rat DNA double-strand break repair genes Ku p70 and Ku p80/XRCC5 and their mRNA expression in various mouse tissues.

The Ku p70 and Ku p80/XRCC5 genes are involved in DNA double-strand break repair and V(D)J recombination, and their gene products are the components of the DNA-dependent protein kinase. We have determined the chromosomal locations of the mouse Ku p70 and Ku p80/XRCC5 genes by both in situ hybridization and molecular linkage analysis: the Ku p70 gene was localized to mouse chromosome 15 and rat chromosome 7, and the Ku p80/XRCC5 gene was localized to mouse chromosome 1 and rat chromosome 9. Both genes were mapped to a region of conserved linkage homology among three species, i.e., the mouse, rat, and human. Molecular linkage analysis using interspecific backcross mice revealed that the murine Ku p70 locus was localized 0.7 cM terminal to D15Mit1 and that the murine Ku p80/XRCC5 locus was 0.7 cM proximal to D1Mit46. To determine the size and tissue transcription specificity of the mouse Ku p70 and Ku p80/XRCC5 mRNA, Northern blot analysis was carried out with six mouse tissues. Each tissue expressed one species of the Ku p70 gene transcript with 2.4 kb and one species of the Ku p80/XRCC5 gene transcript with 2.6 kb. In the latter case, however, the brain showed two sizes of transcript, 2.6 and 2.9 kb.

Animals↗
PubMed1998

Abnormal DNA damage-inducible protein in cells from Sjögren's syndrome patients.

Antinuclear antibodies are commonly found in patients with Sjögren's syndrome. It has been suggested that the development of antinuclear antibodies depends on the activation of the spliceosome and other transcription-related subcellular particles, some of which have recently been shown also to function in DNA-modifying processes, such as DNA repair and V(D)J recombination. These observations add weight to a previously proposed model for the aetiology of Sjögren's syndrome. This includes the abnormal processing of the T-cell receptor and immunoglobulin genes. To test this hypothesis further, the present study on DNA-modifying proteins in Sjögren's syndrome was initiated. Gel-shift experiments using protein extracted from UV-treated Sjögren cells provided evidence of high molecular weight DNA-binding protein in six out of 12 Sjögren patients studied (but not among seven healthy controls). Some Sjögren sera displayed antibodies to protein extracts from cells treated with psoralen plus UVA radiation. These results indicate an abnormal DNA damage-inducible response in Sjögren's syndrome. It may therefore be concluded that alterations in nuclear protein may play a role in the aetiology of Sjögren's syndrome.

Antibodies, Antinuclear↗
PubMed1998

Ku70/Ku80 protein complex inhibits the binding of nucleotide excision repair proteins on linear DNA in vitro.

We have previously reported that the incision efficiency of the nucleotide excision repair (NER) reaction measured in vitro with cell-free human protein extracts was reduced by up to 80% on a linearized damaged plasmid DNA substrate when compared to supercoiled damaged DNA. The inhibition stemed from the presence of the DNA-end binding Ku70/Ku80 heterodimer which is the regulatory subunit of the DNA-dependent protein kinase (DNA-PK). Here, the origin of the repair inhibition was assessed by a new in vitro assay in which circular or linear plasmid DNA, damaged or undamaged, was quantitatively adsorbed on sensitized microplate wells. The binding of two NER proteins, XPA and p62-TFIIH, indispensable for the incision step of the reaction, was quantified either directly in an ELISA-like reaction in the wells with specific antibodies or in Western blotting experiments on the DNA-bound fraction. We report a dramatic inhibition of XPA and p62-TFIIH association with UVC photoproducts on linear DNA. XPA and p62-TFIIH binding to DNA damage was regained when the reaction was performed with extracts lacking Ku activity (extracts from xrs6 rodent cells) whereas addition of purified human Ku complex to these extracts restored the inhibition. Despite the fact that DNA-PK was active during the NER reaction, the mechanism of inhibition relied on the sole Ku complex, since mutant protein extracts lacking the catalytic DNA-PK subunit (extracts from the human M059J glioma cells) exhibited a strong binding inhibition of XPA and p62-TFIIH proteins on linear damaged DNA, identical to the inhibition observed with the DNA-PK+ control extracts (from M059K cells).

Animals↗
PubMed2000

Mass spectral analysis of a protein complex using single-chain antibodies selected on a peptide target: applications to functional genomics.

Genome projects are identifying an ever-increasing number of genes, accelerating the need for reagents to study the expression of these genes and elucidate the function and cellular location of the gene products. Our goal was to develop a strategy to allow human single-chain variable fragment (scFv) antibodies to be used for these endeavors. A library containing 7x10(9) individual variants was displayed by bacteriophage and selected against a biotinylated peptide corresponding to the C-terminal 15 amino acid residues of Ku86, one component of a heterodimer involved in double-stranded DNA break repair. Four unique scFv antibodies were recovered that not only recognized the selected peptide, but also the intact protein. Three of the scFv antibodies were expressed in soluble form and recognized Ku86 by Western analysis. The affinity of one of the scFv antibodies for Ku86 was 16 nM as measured by BIAcore analysis. scFv immunoprecipitation of Ku86 also isolated the other component of the heterodimer, Ku70, as determined by Western analysis and mass spectrometry. These results demonstrate the utility of scFv antibodies as invaluable reagents for functional genomics.

Antibodies↗
PubMed2002

Analysis of the kinetic and equilibrium binding of Ku protein to DNA.

The loading of Ku onto a DNA end in a double-strand DNA break is thought to be one of the first steps in the non-homologous DNA end joining (NHEJ) pathway, giving it an essential role in the maintenance of genomic integrity. The binding of Ku to DNA is complicated since DNA can accommodate multiple Ku subunits, which can translocate on the DNA strand. Furthermore, Ku may exhibit cooperativity in the loading process. Therefore, simple one- to-one kinetic models are unable to adequately simulate the process. However, through the use of computer simulation and curve-fitting, we are able to provide a comprehensive mechanistic model and rate constants that closely approximate experimental data for DNA molecules that bind one, two, and three Ku molecules under both kinetic and equilibrium conditions. The model obtains a best fit with Ku having a roughly seven-fold preference to bind to DNA ends rather than internal positions and is consistent with Ku having a strong preference of which face of the protein loads onto the DNA end.

Animals↗
PubMed1999

A novel neuron-specific DNA end-binding factor in the murine brain.

To characterize the distribution of transcription factor AP-1 and YY1 DNA-binding activities in the rat brain, the labeled target oligonucleotides were loaded on brain sections and after incubation and washing, the residual signal was registered by autoradiography. The binding was predominantly associated with neurons and was regionally specific with highest levels in the cerebellum, hippocampus, and piriform cortex. The identified binding factor was not, however, sequence-specific, but apparently recognized DNA ends and was activated by long double-stranded DNA. UV cross-linking identified the molecular mass of the factor to be about 80 kDa. The factor was not found in soluble brain extracts, suggesting its association with membranes or the nuclear matrix. Despite apparent similarities with Ku protein, which targets DNA-ends, the DNA end-binding activity was present in brains of Ku86- and Ku70-deficient mice. Since DNA end-binding factors are generally involved in DNA repair, the same function may be suggested for the novel factor identified in the present study.

Animals↗
PubMed1997

Essential and perilous: V(D)J recombination and DNA damage checkpoints in lymphocyte precursors.

V(D)J recombination generates a diverse array of antigen-binding specificities, but breakage and re-joining of DNA segments have grave implications for the maintenance of genomic stability and oncogenic risk. Exposure of eukaryotic cells to genotoxic agents activates a DNA damage checkpoint that induces cell-cycle arrest and DNA repair, or apoptosis. We discuss several lines of evidence implicating DNA-dependent protein kinase (DNA-PK), and the gene mutated in ataxia telangiectasia (ATM), two mammalian homologues of yeast DNA damage-checkpoint genes, in regulating the response to intrinsic DNA damage that occurs during V(D)J recombination.

Amino Acid Sequence↗
PubMed2002

Effect of Ku80 depletion on the preintegrative steps of HIV-1 replication in human cells.

To gain new insights regarding the role of Ku, the DNA-PK DNA-binding component, during lentiviral DNA integration, we have investigated the HIV-1 replication in Ku80-depleted human cells. CEM4fx cells underexpressing the Ku80 factor were selected after transduction by a retroviral vector expressing the Ku80 full-length antisense sequence. De novo infection experiment with NL4.3 HIV-1 strain led to the observation that the viral replication was delayed in the Ku80-depleted cells. Early events of the replicative cycle, including nuclear import of the viral DNA, were not affected. In contrast, the formation of the 2-LTR circles was impaired, thus demonstrating the implication of Ku in HIV-1 DNA circularization, for the first time in human cells. Furthermore, the detection of integrated proviruses by an Alu-LTR-nested PCR amplification method was affected in cells underexpressing Ku80. These results suggest that this factor may also be involved in the mechanisms leading to the stable establishment of HIV-1 provirus.

Antigens, Nuclear↗
PubMedDate not supplied

Multiple functional enhancer motifs of rat ribosomal gene.

Previous studies from this laboratory have characterized a 174 bp enhancer element which is located 2 kb upstream of the initiation site. Half of the enhancer action is controlled by a 37 bp element at the 3' end of the 174 bp region. We now report that a 43 bp adjacent domain which is located upstream of the 37 bp element constitutes an additional motif of the rDNA enhancer. When the plasmid consisting of the 43 bp DNA upstream of the rDNA core promoter was transcribed in a fractionated rat tumor cell extract (fraction DE-B), transcription of rDNA was augmented 4 fold. Electrphoretic mobility shift and DNAase I footprinting analyses showed that the purified 37 bp enhancer (E1)-binding protein, (E1BF) not only interacted with the enhancer motif E1 but also interacted with the neighbouring 43 bp enhancer domain E2. The specificity of the binding was demonstrated by competition with unlabelled 37 bp and 43 bp fragment and lack of competition with nonspecific DNAs in the mobility shift assay. These studies have shown that a single pol I transcription factor can bind to multiple enhancer domains with no significant sequence homologies and such multiple interactions may result in maximal transcription of ribosomal gene from the core promoter.

Animals↗