Hypertonic treatment inhibits radiation-induced nuclear translocation of the Ku proteins G22p1 (Ku70) and Xrcc5 (Ku80) in rat fibroblasts

The effects of X irradiation and hypertonic treatment with 0.5 M NaCl on the subcellular localization of the Ku proteins G22p1 (also known as Ku70) and Xrcc5 (also known as Ku80) in rat fibroblasts with normal radiosensitivity were examined using confocal laser microscopy and immunoblotting. Although these proteins were observed mainly in the nuclei of human fibroblasts, approximately 80% of the intensities of immunofluorescence from both G22p1 and Xrcc5 was observed in the cytoplasm of rat fibroblasts. When the rat cells were X-irradiated with 4 Gy, the intensities of the fluorescence derived from G22p1 and Xrcc5 in the nuclei increased from 20% to 50% of the total cellular fluorescence intensity at 20 min postirradiation. No significant differences were observed between the total intensities of the cellular fluorescence from the proteins in unirradiated and irradiated rat fibroblasts. The results showed that the proteins were translocated from the cytoplasm to the nucleus in the rat cells after X irradiation. The nuclear translocation of the proteins from the cytoplasm was inhibited by hypertonic treatment of the cells with 0.5 M NaCl for 20 min, which inhibits the fast repair process of potentially lethal damage (PLD). When the rat cells were treated with 0.5 M NaCl immediately after X irradiation, the repair of DNA DSBs was inhibited. The surviving fraction was approximately 60% of that of irradiated cells that were not treated with 0.5 M NaCl. The surviving fraction increased with incubation time in the growth medium before treatment with NaCl. The proportions of the intensities of fluorescence from G22p1 in the nuclei of X-irradiated cells also increased from 20% to 50% with increasing interval between X irradiation and treatment with NaCl. These results suggest that nuclear translocation of G22p1 and Xrcc5 is important for the fast repair process of PLD in rat cells.     

Reprogramming cellular events by poly(ADP-ribose)-binding proteins

Poly(ADP-ribosyl)ation is a posttranslational modification catalyzed by the poly(ADP-ribose) polymerases (PARPs). These enzymes covalently modify glutamic, aspartic and lysine amino acid side chains of acceptor proteins by the sequential addition of ADP-ribose (ADPr) units. The poly(ADP-ribose) (pADPr) polymers formed alter the physico-chemical characteristics of the substrate with functional consequences on its biological activities. Recently, non-covalent binding to pADPr has emerged as a key mechanism to modulate and coordinate several intracellular pathways including the DNA damage response, protein stability and cell death. In this review, we describe the basis of non-covalent binding to pADPr that has led to the emerging concept of pADPr-responsive signaling pathways. This review emphasizes the structural elements and the modular strategies developed by pADPr-binding proteins to exert a fine-tuned control of a variety of pathways. Poly(ADP-ribosyl)ation reactions are highly regulated processes, both spatially and temporally, for which at least four specialized pADPr-binding modules accommodate different pADPr structures and reprogram protein functions. In this review, we highlight the role of well-characterized and newly discovered pADPr-binding modules in a diverse set of physiological functions.     

mRNA-specific regulation of translation by poly(A)-binding proteins

The regulation of translation has emerged as a major determinant of gene expression and is critical for both normal cellular function and the development of disease. Numerous studies have highlighted the diverse, and sometimes related, mechanisms which underlie the regulation of global translation rates and the translational control of specific mRNAs. In the present paper, we discuss the emerging roles of the basal translation factor PABP [poly(A)-binding protein] in mRNA-specific translational control in metazoa which suggest that PABP function is more complex than first recognized.     

Epitopes of the p70 and p80 (Ku) lupus autoantigens.

High titer autoantibodies to the Ku Ag, a DNA-protein complex containing 70- and approximately 80-kDa protein subunits (p70 and p80, respectively), are found in sera of certain patients with systemic lupus erythematosus and related disorders. Autoepitopes of the Ku Ag were identified and partially characterized by expressing fragments of the p70 and p80 cDNA as fusion proteins in bacteria. Systemic lupus erythematosus sera reacted on immunoblots with at least three epitopes of p70 (amino acids 560-609, 506-535, and 115-467), and three epitopes of p80 (amino acids 682-732, 558-681, and 1-374). These six antigenic regions had distinct amino acid sequences, and were also immunologically distinct, as determined by using immunoaffinity-purified auto-antibodies to particular epitopes. Detailed mapping of the strongly antigenic region near the C terminus of p70 revealed a complex conformational or discontinuous epitope, the antigenicity of which was abolished by deleting either amino acids 560-571 or 601-609. The C terminus of p80 may also contain a discontinuous or conformational epitope(s). Although only some sera reacted with p70 or p80 on immunoblots, all sera that immunoprecipitated the native Ku complex reacted with native Ku by ELISA, and inhibited the binding of mAb directed at epitopes of native Ku. Taken together, these studies indicate that anti-Ku autoantibodies target a diversity of independent epitopes located on p70, p80, and the intact Ku complex, and that a significant portion of the autoantibodies in most patients' sera is directed against conformational/discontinuous epitopes.     

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.     

Expression and chromosome location of hamster Ku70 and Ku80

Ku proteins play an important role in DNA double-strand break (DSB) repair, chromosome maintenance, and growth regulation. To understand the fundamental characteristics of Ku proteins, we examined the electrophoretic mobility and expression of hamster Ku70 and Ku80 and determined the chromosome locations of their genes. The electrophoretic mobility of hamster Ku proteins are different from that of human Ku proteins. No significant changes in the quantity of Ku proteins were observed in CHO-K1 cells treated with 10 Gy of ionizing radiation, suggesting that both proteins are expressed constitutively in amounts adequate to repair DNA DSBs. The chromosome locations of the Ku genes were determined by direct R-banding fluorescence in situ hybridization. The Ku70 gene was localized to Syrian hamster chromosome 4qa4.1--> qa4.2 and Chinese hamster chromosome 2p3.1, and the Ku80 gene was localized to Syrian hamster chromosome 4qb5--> qb6.1 and Chinese hamster chromosome 2p3.5-->p3.6. These results provide clues to the biological functions of Ku, as well as useful information for constructing comparative chromosome maps between hamsters and other mammalian species, including human, mouse, and rat.     

Regulation of homologous integration in yeast by the DNA repair proteins Ku70 and RecQ

The product of the BLM gene, which is mutated in Bloom syndrome in humans, and the Saccharomyces cerevisiae protein Sgs1 are both homologous to the Escherichia coli DNA helicase RecQ, and have been shown to be involved in the regulation of homologous recombination. Mutations in these genes result in genome instability because they increase the incidence of deletions and translocations. We present evidence for a genetic interaction between SGS1 and YKU70, which encodes the S. cerevisiae homologue of the human DNA helicase Ku70. In a yku70 mutant background, sgs1 mutations increased sensitivity to DNA breakage induced either by treatment with camptothecin or by the expression of the restriction enzyme EcoRI. The yku70 mutation caused a fourfold increase in the rate of double-strand break (DSB)-induced target integration as that seen in the sgs1 mutant. The combination of yku70 and sgs1 mutations additively increased the rate of the targeted integration, and this effect was completely suppressed by deletion of RAD51. Interestingly, an extra copy of YKU70 partially suppressed the increase in targeted integration seen in the sgs1 single mutant. These results suggest that Yku70 modulates the repair of DSBs associated with homologous recombination in a different way from Sgs1, and that the inactivation of RecQ and Ku70 homologues may enhance the frequency of gene targeting in higher eukaryotes.     

Selected nuclear matrix proteins are targets for poly(ADP-ribose)-binding

Recent evidence suggests that poly(ADP-ribose) may take part in DNA strand break signalling due to its ability to interact with and affect the function of specific target proteins. Using a poly(ADP-ribose) blot assay, we have found that several nuclear matrix proteins from human and murine cells bind ADP-ribose polymers with high affinity. The binding was observed regardless of the procedure used to isolate nuclear matrices, and it proved resistant to high salt concentrations. In murine lymphoma LY-cell cultures, the spontaneous appearance of radiosensitive LY-S sublines was associated with a loss of poly(ADP-ribose)-binding of several nuclear matrix proteins. Because of the importance of the nuclear matrix in DNA processing reactions, the targeting of matrix proteins could be an important aspect of DNA damage signalling via the poly ADP-ribosylation system. J. Cell. Biochem. 70:596–603. © 1998 Wiley-Liss, Inc.     

Non-inositol 1,4,5-trisphosphate (IP3) receptor IP3-binding proteins

Conventionally, myo-D-inositol 1, 4,5-trisphosphate (IP₃) is thought to exert its second messenger effects through the gating of IP₃R Ca²⁺ release channels, located in Ca²⁺-storage organelles like the endoplasmic reticulum. However, there is considerable indirect evidence to support the concept that IP₃ might interact with other, non-IP₃R proteins within cells. To explore this possibility further, the Protein Data Bank was searched using the term “IP3”. This resulted in the retrieval of 203 protein structures, the majority of which were members of the IP₃R/ryanodine receptor superfamily of channels. Only 49 of these structures were complexed with IP₃. These were inspected for their ability to interact with the carbon-1 phosphate of IP₃, since this is the least accessible phosphate group of its precursor, phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂). This reduced the number of structures retrieved to 35, of which 9 were IP₃Rs. The remaining 26 structures represent a diverse range of proteins, including inositol-lipid metabolizing enzymes, signal transducers, PH domain containing proteins, cytoskeletal anchor proteins, the TRPV4 ion channel, a retroviral Gag protein and fibroblast growth factor 2. Such proteins may impact on IP₃ signalling and its effects on cell-biology. This represents an area open for exploration in the field of IP₃ signalling.     

Evidence for Ku70/Ku80 association with full-length RAG1

Antigen receptor genes are assembled by a site-specific DNA rearrangement process called V(D)J recombination. This process proceeds through two distinct phases: a cleavage phase in which the RAG1 and RAG2 proteins introduce DNA double-strand breaks at antigen receptor gene segments, and a joining phase in which the resulting DNA breaks are processed and repaired via the non-homologous end-joining (NHEJ) repair pathway. Genetic and biochemical evidence suggest that the RAG proteins play an active role in guiding the repair of DNA breaks introduced during V(D)J recombination to the NHEJ pathway. However, evidence for specific association between the RAG proteins and any of the factors involved in NHEJ remains elusive. Here we present evidence that two components of the NHEJ pathway, Ku70 and Ku80, interact with full-length RAG1, providing a biochemical link between the two phases of V(D)J recombination.     

Oxidative stress induces nuclear loss of DNA repair proteins Ku70 and Ku80 and apoptosis in pancreatic acinar AR42J cells

Cell death linked to oxidative DNA damage has been implicated in acute pancreatitis. The severe DNA damage, which is beyond the capacity of the DNA repair proteins, triggers apoptosis. It has been hypothesized that oxidative stress may induce a decrease in the Ku70 and Ku80 levels and apoptosis in pancreatic acinar cells. In this study, it was found that oxidative stress caused by glucose oxidase (GO) acting on beta-d-glucose, glucose/glucose oxidase (G/GO), induced slight changes in cytoplasmic Ku70 and Ku80 but drastically induced a decrease in nuclear Ku70 and Ku80 both time- and concentration-dependently in AR42J cells. G/GO induced apoptosis determined by poly(ADP-ribose) polymerase cleavage, an increase in expression of p53 and Bax, and a decrease in Bcl-2 expression. G/GO-induced apoptosis was in parallel with the loss of nuclear Ku proteins in AR42J cells. Caspase-3 inhibitor prevented G/GO-induced nuclear Ku loss and cell death. G/GO did not induce apoptosis in the cells transfected with either the Ku70 or Ku80 expression gene but increased apoptosis in those transfected with the Ku dominant negative mutant. Pulse and pulse-chase results show that G/GO induced Ku70 and Ku80 syntheses, even though Ku70 and Ku80 were degraded both in cytoplasm and nucleus. G/GO-induced decrease in Ku binding to importin alpha and importin beta reflects possible modification of nuclear import of Ku proteins. The importin beta level was not changed by G/GO. These results demonstrate that nuclear decrease in Ku70 and Ku80 may result from the decrease in Ku binding to nuclear transporter importins and the degradation of Ku proteins. The nuclear loss of Ku proteins may underlie the mechanism of apoptosis in pancreatic acinar cells after oxidative stress.     

Identification of poly(ADP-ribose)polymerase-1 and Ku70/Ku80 as transcriptional regulators of S100A9 gene expression

Background  

S100 proteins, a multigenic family of non-ubiquitous cytoplasmic Ca²⁺-binding proteins, have been linked to human pathologies in recent years. Dysregulated expression of S100 proteins, including S100A9, has been reported in the epidermis as a response to stress and in association with neoplastic disorders. Recently, we characterized a regulatory element within the S100A9 promotor, referred to as MRE that drives the S100A9 gene expression in a cell type-specific, activation- and differentiation-dependent manner (Kerkhoff et al. (2002) J. Biol. Chem. 277, 41879–41887).     

Nucleotide sequence and genomic structure analyses of the p70 subunit of the human Ku autoantigen: evidence for a family of genes encoding Ku (p70)-related polypeptides

cDNA encoding the p70 polypeptide subunit of the human Ku autoantigen was isolated. In vitro expression analysis of the cDNA demonstrates that it encodes the entire open reading frame. Nucleotide sequence analysis and comparison to other previously described sequences indicate the existence of several single-nucleotide and amino acid polymorphisms. Southern blot analyses demonstrate the presence of multiple copies of homologous DNA sequences in the human genome. These data support the hypothesis that multiple genes encode a family of Ku(p70)-related polypeptides.     

Somatomedin (insulin-like growth factors)-binding proteins. Molecular forms and regulation

The insulin-like growth factor (IGF)-binding proteins present in the human serum and various biological media have been characterized by several methods: gel filtration, sucrose gradient sedimentation, polyacrylamide gel electrophoresis and chromatofocusing. Several forms have been identified with molecular weights of approximately 42,000, 39,000, 34,000, 30,000 and 24,000 daltons. Results of competitive binding studies suggest that the different forms of binding proteins have different affinities for IGF-I and IGF-II. The influence of various hormones and pathophysiological conditions on the biosynthesis of the binding proteins has been investigated.     

Interaction of human Ku70 with TRF2

Ku, a heterodimer of 70- and 80-kDa subunits, plays a general role in the metabolism of DNA ends in eukaryotic cells, including double-strand DNA break repair, V(D)J recombination, and maintenance of telomeres. We have utilized the yeast two-hybrid system to identify Ku70-interacting proteins other than Ku80. Two reactive clones were found to encode the dimerization domain of TRF2, a mammalian telomeric protein that binds to duplex TTAGGG repeats at chromosome ends. This interaction was confirmed using bacterial fusion proteins and co-immunoprecipitations from eukaryotic cells overexpressing TRF2. The transfected TFR2 colocalized with Ku70.