Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Ku Autoantigen”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 487 records · Page 27Linked to original sources
PubMed2000

Ku entry into DNA inhibits inward DNA transactions in vitro.

Association of the DNA end-binding Ku70/Ku80 heterodimer with the 460-kDa serine/threonine kinase catalytic subunit forms the DNA-dependent protein kinase (DNA-PK) that is required for double-strand break repair by non-homologous recombination in mammalian cells. Recently, we have proposed a model in which the kinase activity is required for translocation of the DNA end-binding subunit Ku along the DNA helix when DNA-PK assembles on DNA ends. Here, we have questioned the consequences of Ku entry into DNA on local DNA processes by using human nuclear cell extracts incubated in the presence of linearized plasmid DNA. As two model processes, we have chosen nucleotide excision repair (NER) of UVC DNA lesions and transcription from viral promoters. We show that although NER efficiency is strongly reduced on linear DNA, it can be fully restored in the presence of DNA-PK inhibitors. Simultaneously, the amount of NER proteins bound to the UVC-damaged linear DNA is increased and the amount of Ku bound to the same DNA molecules is decreased. Similarly, the poor transcription efficiency exhibited by viral promoters on linear DNA is enhanced in the presence of DNA-PK inhibitor concentrations that prevent Ku entry into the DNA substrate molecule. The present results show that DNA-PK catalytic activity can regulate DNA transactions including transcription in the vicinity of double-strand breaks by controlling Ku entry into DNA.

Androstadienes↗
PubMed2000

Human Ku antigen tightly binds and stabilizes a tetrahelical form of the Fragile X syndrome d(CGG)n expanded sequence.

Hairpin and tetrahelical structures of a d(CGG)(n) sequence in the FMR1 gene have been implicated in its expansion in fragile X syndrome. The identification of tetraplex d(CGG)(n) destabilizing proteins (Fry, M., and Loeb, L. A.(1999) J. Biol. Chem. 274, 12797-12803; Weisman-Shomer, P., Naot, Y., and Fry, M. (2000) J. Biol. Chem. 275, 2231-2238) suggested that proteins might modulate d(CGG)(n) folding and aggregation. We assayed human TK-6 lymphoblastoid cell extracts for d(CGG)(8) oligomer binding proteins. The principal binding protein was identified as Ku antigen by its partial amino acid sequence and antigenicity. The purified 88/75-kDa heterodimeric Ku bound with similar affinities (K(d) approximately 1. 8-10.2 x 10(-9) mol/liter) to double-stranded d(CGG)(8).d(CCG)(8), hairpin d(CGG)(8), single-stranded d(CII)(8), or tetraplex structures of telomeric or IgG switch region sequences. However, Ku associated more tightly with bimolecular G'2 tetraplex d(CGG)(8) (K(d) approximately 0.35 x 10(-9) mol/liter). Binding to Ku protected G'2 d(CGG)(8) against nuclease digestion and impeded its unwinding by the tetraplex destabilizing protein qTBP42. Stabilization of d(CGG)(n) tetraplex domains in FMR1 by Ku or other proteins might promote d(CGG) expansion and FMR1 silencing.

Amino Acid Sequence↗
PubMed2001

Requirements for the nucleolytic processing of DNA ends by the Werner syndrome protein-Ku70/80 complex.

Werner syndrome (WS) is an inherited disease characterized by premature onset of aging, increased cancer incidence, and genomic instability. The WS gene encodes a protein with helicase and exonuclease activities. Our previous studies indicated that the Werner syndrome protein (WRN) interacts with Ku, a heterodimeric factor of 70- and 80-kDa subunits implicated in the repair of double strand DNA breaks. Moreover, we demonstrated that Ku70/80 strongly stimulates and alters WRN exonuclease activity. In this report, we investigate further the association between WRN and Ku70/80. First, using various WRN deletion mutants we show that 50 amino acids at the amino terminus are required and sufficient to interact with Ku70/80. In addition, our data indicate that the region of Ku80 between amino acids 215 and 276 is necessary for binding to WRN. Then, we show that the amino-terminal region of WRN from amino acid 1 to 388, which comprise the exonuclease domain, can be efficiently stimulated by Ku to degrade DNA substrates, indicating that the helicase domain and the carboxyl-terminal tail are not required for the stimulatory process. Finally, using gel shift assays, we demonstrate that Ku recruits WRN to DNA. Taken together, these results suggest that Ku-mediated activation of WRN exonuclease activity may play an important role in a cellular pathway that requires processing of DNA ends.

Adenosine Triphosphate↗
PubMed2000

Human Ku70 interacts with heterochromatin protein 1alpha.

Ku is involved in the metabolism of DNA ends, DNA repair, and the maintenance of telomeres. It consists of a heterodimer of 70- and 80-kDa subunits. Recently we have demonstrated that Ku70 interacted with TRF2, a mammalian telomere-binding protein. Using the same yeast two-hybrid screening system, we now show that Ku70 also interacts with heterochromatin protein 1alpha (HP1alpha), a protein known to be associated with telomeres as well as heterochromatin. HP1 is a suppressor of the position effect variegation in Drosophila and acts as a transcriptional suppressor in mammalian cells. The interaction with Ku70 in the two-hybrid system was confirmed by a glutathione S-transferase pull-down study using bacterial recombinant proteins in vitro. The interaction was also reproduced in vivo in HeLa cells, where endogenous Ku70 coimmunoprecipitated with HP1alpha. This interaction was more effective in acidic pH and weakened considerably as the pH of the reaction buffer was elevated up to 7.5. Ku80 did not interact with HP1alpha directly. The interaction domains of Ku70 and HP1alpha included the Leu-Ser repeat (amino acids 200-385) and the chromo shadow domain, respectively. Ku70 was largely colocalized with transfected HP1alpha but not with a C-terminal deletion mutant, HP1alpha(Delta)C. In contrast to HP1alpha, Ku70 did not repress transcriptional activity of the reporter gene when tethered to DNA after transfection to mammalian cells. The implication of this interaction is discussed.

Antigens, Nuclear↗
PubMed2001

Accurate in vitro end joining of a DNA double strand break with partially cohesive 3'-overhangs and 3'-phosphoglycolate termini: effect of Ku on repair fidelity.

To examine determinants of fidelity in DNA end joining, a substrate containing a model of a staggered free radical-mediated double-strand break, with cohesive phosphoglycolate-terminated 3'-overhangs and a one-base gap in each strand, was constructed. In extracts of Xenopus eggs, human lymphoblastoid cells, hamster CHO-K1 cells, and a Chinese hamster ovary (CHO) derivative lacking the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), the predominant end joining product was that corresponding to accurate restoration of the original sequence. In extracts of the Ku-deficient CHO derivative xrs6, a shorter product, consistent with 3' --> 5' resection before ligation, was formed. Similar results were seen for a substrate with 5'-overhangs and recessed 3'-phosphoglycolate ends. Supplementation of the xrs6 extracts with purified Ku restored accurate end joining. In Xenopus and human extracts, but not in hamster extracts, gap filling and ligation were blocked by wortmannin, consistent with a requirement for DNA-PKcs activity. The results suggest a Ku-dependent pathway, regulated by DNA-PKcs, that can accurately restore the original DNA sequence at sites of free radical-mediated double-strand breaks, by protecting DNA termini from degradation and maintaining the alignment of short partial complementarities during gap filling and ligation.

Androstadienes↗
PubMed2001

Distinct roles for Ku protein in transcriptional reinitiation and DNA repair.

Transcriptional reinitiation is a distinct phase of the RNA polymerase II transcription cycle. Prior work has shown that reinitiation is deficient in nuclear extracts from Chinese hamster ovary cells lacking the 80-kDa subunit of Ku, a double-strand break repair protein, and that activity is rescued by expression of the corresponding cDNA. We now show that Ku increases the amount or availability of a soluble factor that is limiting for reinitiation, that the factor increases the number of elongation complexes associated with the template at all times during the reaction, and that the factor itself does not form a tight complex with DNA. The factor may consist of a preformed complex of transcription proteins that is stabilized by Ku. A Ku mutant, lacking residues 687-728 in the 80-kDa subunit, preferentially suppresses transcription in Ku-containing extracts, suggesting that Ku interacts directly with proteins required for reinitiation. The Ku mutant functions normally in a DNA end-joining system, indicating that the functions of Ku in transcription and repair are genetically separable. Based on our results, we present a model in which Ku is capable of undergoing a switch between a transcription factor-associated and a repair-active state.

Animals↗
PubMed2001

Dimerization and nuclear localization of ku proteins.

Ku, a heterodimer of Ku70 and Ku80, plays a key role in multiple nuclear processes, e.g. DNA repair, chromosome maintenance, and transcription regulation. Heterodimerization is essential for Ku-dependent DNA repair in vivo, although its role is poorly understood. Some lines of evidence suggest that heterodimerization is required for the stabilization of Ku70 and Ku80. Here we show that the heterodimerization of these Ku subunits is important for their nuclear entry. When transfected into Ku-deficient xrs-6 cells, exogenous Ku70 and Ku80 tagged with green fluorescent protein accumulated into the nucleus, whereas each nuclear localization signal (NLS)-dysfunctional mutant was undetectable in the nucleus, supporting the idea that each Ku can translocate to the nucleus through its own NLS. On the other hand, the nuclear accumulation of each NLS-dysfunctional mutant was markedly enhanced by the presence of an exogenous wild-type counterpart. In Ku-expressing HeLa cells, each NLS-dysfunctional mutant, as well as wild-type Ku70 and Ku80, was still detectable in the nucleus, whereas the double mutant of each Ku subunit with decreased functions of both nuclear targeting and dimerization was undetectable in the nucleus. Our results indicate that each Ku subunit can translocate to the nucleus not only through its own NLS but also through heterodimerization with each other.

Animals↗
PubMed2001

A possible role of Ku in mediating sequential repair of closely opposed lesions.

One of the hallmarks of ionizing radiation exposure is the formation of clustered damage that includes closely opposed lesions. We show that the Ku70/80 complex (Ku) has a role in the repair of closely opposed lesions in DNA. DNA containing a dihydrouracil (DHU) close to an opposing single strand break was used as a model substrate. It was found that Ku has no effect on the enzymatic activity of human endonuclease III when the substrate DNA contains only DHU. However, with DNA containing a DHU that is closely opposed to a single strand break, Ku inhibited the nicking activity of human endonuclease III as well as the amount of free double strand breaks induced by the enzyme. The inhibition on the formation of a free double strand break by Ku was found to be much greater than the inhibition of human endonuclease III-nicking activity by Ku. Furthermore, there was a concomitant increase in the formation of Ku-DNA complexes when endonuclease III was present. Similar results were also observed with Escherichia coli endonuclease III. These results suggest that Ku reduces the formation of endonuclease III-induced free double strand breaks by sequestering the double strand breaks formed as a Ku-DNA complex. In doing so, Ku helps to avoid the formation of the intermediary free double strand breaks, possibly helping to reduce the mutagenic event that might result from the misjoining of frank double strand breaks.

Antigens, Nuclear↗
PubMed2001

Protein phosphatases regulate DNA-dependent protein kinase activity.

DNA-dependent protein kinase (DNA-PK) is a complex of DNA-PK catalytic subunit (DNA-PKcs) and the DNA end-binding Ku70/Ku80 heterodimer. DNA-PK is required for DNA double strand break repair by the process of nonhomologous end joining. Nonhomologous end joining is a major mechanism for the repair of DNA double strand breaks in mammalian cells. As such, DNA-PK plays essential roles in the cellular response to ionizing radiation and in V(D)J recombination. In vitro, DNA-PK undergoes phosphorylation of all three protein subunits (DNA-PK catalytic subunit, Ku70 and Ku80) and phosphorylation correlates with inactivation of the serine/threonine protein kinase activity of DNA-PK. Here we show that phosphorylation-induced loss of the protein kinase activity of DNA-PK is restored by the addition of the purified catalytic subunit of either protein phosphatase 1 or protein phosphatase 2A (PP2A) and that this reactivation is blocked by the potent protein phosphatase inhibitor, microcystin. We also show that treating human lymphoblastoid cells with either okadaic acid or fostriecin, at PP2A-selective concentrations, causes a 50-60% decrease in DNA-PK protein kinase activity, although the protein phosphatase 1 activity in these cells was unaffected. In vivo phosphorylation of DNA-PKcs, Ku70, and Ku80 was observed when cells were labeled with [(32)P]inorganic phosphate in the presence of the protein phosphatase inhibitor, okadaic acid. Together, our data suggest that reversible protein phosphorylation is an important mechanism for the regulation of DNA-PK protein kinase activity and that the protein phosphatase responsible for reactivation in vivo is a PP2A-like enzyme.

Alkenes↗
PubMed2001

The binding of Ku antigen to homeodomain proteins promotes their phosphorylation by DNA-dependent protein kinase.

The Ku antigen (70- and 80-kDa subunits) is a regulatory subunit of DNA-dependent protein kinase (DNA-PK) that promotes the recruitment of the catalytic subunit of DNA-PK (DNA-PKcs) to DNA ends and to specific DNA sequences from which the kinase is activated. Ku and DNA-PKcs plays essential roles in double-stranded DNA break repair and V(D)J recombination and have been implicated in the regulation of specific gene transcription. In a yeast two-hybrid screen of a Jurkat T cell cDNA library, we have identified a specific interaction between the 70-kDa subunit of Ku heterodimer and the homeodomain of HOXC4, a homeodomain protein expressed in the hematopoietic system. Unexpectedly, a similar interaction with Ku was observed for several additional homeodomain proteins including octamer transcription factors 1 and 2 and Dlx2, suggesting that specific binding to Ku may be a property shared by many homeodomain proteins. Ku-homeodomain binding was mediated through the extreme C terminus of Ku70 and was abrogated by amino acid substitutions at Lys595/Lys596. Ku binding allowed the recruitment of the homeodomain to DNA ends and dramatically enhanced the phosphorylation of homeodomain-containing proteins by DNA-PK. These results suggest that Ku functions as a substrate docking protein for signaling by DNA-PK to homeodomain proteins from DNA ends.

Adrenocorticotropic Hormone↗
PubMed2001

The three-dimensional structure of the C-terminal DNA-binding domain of human Ku70.

The proteins Ku70 (69.8 kDa) and Ku80 (82.7 kDa) form a heterodimeric complex that is an essential component of the nonhomologous end joining DNA double-strand break repair pathway in mammalian cells. Interaction of Ku with DNA is central for the functions of Ku. Ku70, which is mainly responsible for the DNA binding activity of the Ku heterodimer, contains two DNA-binding domains. We have solved the solution structure of the Ku80-independent DNA-binding domain of Ku70 encompassing residues 536-609 using nuclear magnetic resonance spectroscopy. Residues 536-560 are highly flexible and have a random structure but form specific interactions with DNA. Residues 561-609 of Ku70 form a well defined structure with 3 alpha-helices and also interact with DNA. The three-dimensional structure indicates that all conserved hydrophobic residues are in the hydrophobic core and therefore may be important for structural integrity. Most of the conserved positively charged residues are likely to be critical for DNA recognition. The C-terminal DNA-binding domain of Ku70 contains a helix-extended strand-helix motif, which occurs in other nucleic acid-binding proteins and may represent a common nucleic acid binding motif.

Amino Acid Sequence↗
PubMed2001

Genetic analysis of the DNA-dependent protein kinase reveals an inhibitory role of Ku in late S-G2 phase DNA double-strand break repair.

Two major complementary double-strand break (DSB) repair pathways exist in vertebrates, homologous recombination (HR), which involves Rad54, and non-homologous end-joining, which requires the DNA-dependent protein kinase (DNA-PK). DNA-PK comprises a catalytic subunit (DNA-PKcs) and a DNA-binding Ku70 and Ku80 heterodimer. To define the activities of individual DNA-PK components in DSB repair, we targeted the DNA-PKcs gene in chicken DT40 cells. DNA-PKcs deficiency caused a DSB repair defect that was, unexpectedly, suppressed by KU70 disruption. We have shown previously that genetic ablation of Ku70 confers RAD54-dependent radioresistance on S-G(2) phase cells, when sister chromatids are available for HR repair. To test whether direct interference by Ku70 with HR might explain the Ku70(-/-)/DNA-PKcs(-/-/-) radioresistance, we monitored HR activities directly in Ku- and DNA-PKcs-deficient cells. The frequency of intrachromosomal HR induced by the I-SceI restriction enzyme was increased in the absence of Ku but not of DNA-PKcs. Significantly, abrogation of HR activity by targeting RAD54 in Ku70(-/-) or DNA-PKcs(-/-/-) cells caused extreme radiosensitivity, suggesting that the relative radioresistance seen with loss of Ku70 was because of HR-dependent repair pathways. Our findings suggest that Ku can interfere with HR-mediated DSB repair, perhaps competing with HR for DSB recognition.

Animals↗
PubMed2001

Coordinate action of the helicase and 3' to 5' exonuclease of Werner syndrome protein.

Werner syndrome is a human disorder characterized by premature aging, genomic instability, and abnormal telomere metabolism. The Werner syndrome protein (WRN) is the only known member of the RecQ DNA helicase family that contains a 3' --> 5'-exonuclease. However, it is not known whether both activities coordinate in a biological pathway. Here, we describe DNA structures, forked duplexes containing telomeric repeats, that are substrates for the simultaneous action of both WRN activities. We used these substrates to study the interactions between the WRN helicase and exonuclease on a single DNA molecule. WRN helicase unwinds at the forked end of the substrate, whereas the WRN exonuclease acts at the blunt end. Progression of the WRN exonuclease is inhibited by the action of WRN helicase converting duplex DNA to single strand DNA on forks of various duplex lengths. The WRN helicase and exonuclease act in concert to remove a DNA strand from a long forked duplex that is not completely unwound by the helicase. We analyzed the simultaneous action of WRN activities on the long forked duplex in the presence of the WRN protein partners, replication protein A (RPA), and the Ku70/80 heterodimer. RPA stimulated the WRN helicase, whereas Ku stimulated the WRN exonuclease. In the presence of both RPA and Ku, the WRN helicase activity dominated the exonuclease activity.

Antigens, Nuclear↗
PubMed2001

Ku represses the HIV-1 transcription: identification of a putative Ku binding site homologous to the mouse mammary tumor virus NRE1 sequence in the HIV-1 long terminal repeat.

Ku has been implicated in nuclear processes, including DNA break repair, transcription, V(D)J recombination, and telomere maintenance. Its mode of action involves two distinct mechanisms: one in which a nonspecific binding occurs to DNA ends and a second that involves a specific binding to negative regulatory elements involved in transcription repression. Such elements were identified in mouse mammary tumor virus and human T cell leukemia virus retroviruses. The purpose of this study was to investigate a role for Ku in the regulation of human immunodeficiency virus (HIV)-1 transcription. First, HIV-1 LTR activity was studied in CHO-K1 cells and in CH0-derived xrs-6 cells, which are devoid of Ku80. LTR-driven expression of a reporter gene was significantly increased in xrs-6 cells. This enhancement was suppressed after re-expression of Ku80. Second, transcription of HIV-1 was followed in U1 human cells that were depleted in Ku by using a Ku80 antisense RNA. Ku depletion led to a increase of both HIV-1 mRNA synthesis and viral production compared with the parent cells. These results demonstrate that Ku acts as a transcriptional repressor of HIV-1 expression. Finally, a putative Ku-specific binding site was identified within the negative regulatory region of the HIV-1 long terminal repeat, which may account for this repression of transcription.

3T3 Cells↗
PubMed2002

Mutation in the glucose-6-phosphate dehydrogenase gene leads to inactivation of Ku DNA end binding during oxidative stress.

Glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the oxidative pentose phosphate cycle, regulates the NADPH/NADP(+) ratio in eukaryotic cells. G6PD deficiency is one of the most common mutations in humans and is known to cause health problems for hundreds of millions worldwide. Although it is known that decreased G6PD functionality can result in increased susceptibility to oxidative stress, the molecular targets of this stress are not known. Using a Chinese hamster ovary G6PD-null mutant, we previously demonstrated that exposure to a thiol-specific oxidant, hydroxyethyldisulfide, caused enhanced radiation sensitivity and an inability to repair DNA double strand breaks. We now demonstrate a molecular mechanism for these observations: the direct inhibition of DNA end binding activity of the Ku heterodimer, a DNA repair protein, by oxidation of its cysteine residues. Inhibition of Ku DNA end binding was found to be reversible by treatment of the nuclear extract with dithiothreitol, suggesting that the homeostatic regulation of reduced cysteine residues in Ku is a critical function of G6PD and the oxidative pentose cycle. In summary, we have discovered a new layer of DNA damage repair, that of the functional maintenance of repair proteins themselves. In view of the rapidly escalating number of roles ascribed to Ku, these results may have widespread ramifications.

Animals↗
PubMed2002

Studies on the mode of Ku interaction with DNA.

The Ku heterodimer plays a central role in non-homologous end-joining. The binding of recombinant Ku to DNA has been investigated by dynamic light scattering, double-filter binding, fluorescence spectroscopy, and band shift assays. The hydrodynamic radius of Ku in solution is 5.2 nm and does not change when a 25-bp double-strand DNA (dsDNA) fragment (D25) is added, indicating that only one Ku molecule binds to a 25-bp fragment. The dissociation constant (k(d)) for the binding to D25 is 3.8 +/- 0.9 nm. If both ends of the substrate are closed with hairpin loops, Ku is still able to bind with little change in the k(d). The k(d) is not affected by ATP, Mg(2+), or ionic strength. However, the addition of bovine serum albumin decreases the k(d) by 2-fold. DNA substrates of 50 bp can bind two Ku molecules, whereas three molecules are bound to a 75-bp substrate. Data analysis with the Hill equation yields a value of the Hill coefficient (n) close to 1, and the k(d) values for the binding of Ku to both ends of these substrates are the same. Thus, we demonstrate that there is no cooperative interaction among the Ku heterodimers binding longer substrates.

Adenosine Triphosphate↗
PubMed2002

Ku is important for telomere maintenance, but not for differential expression of telomeric VSG genes, in African trypanosomes.

Trypanosome antigenic variation, involving differential expression of variant surface glycoprotein (VSG) genes, has a strong association with telomeres and with DNA recombination. All expressed VSGs are telomeric, and differential activation involves recombination into the telomeric environment or silencing/activation of subtelomeric promoters. A number of pathogen contingency gene systems associated with immune evasion involve telomeric loci, which has prompted speculation that chromosome ends provide conditions conducive for the operation of rapid gene switching mechanisms. Ku is a protein associated with eukaryotic telomeres that is directly involved in DNA recombination and in gene silencing. We have tested the hypothesis that Ku in trypanosomes is centrally involved in differential VSG expression. We show, via the generation of null mutants, that trypanosome Ku is closely involved in telomere length maintenance, more so for a transcriptionally active than an inactive telomere, but exhibits no detectable influence on DNA double strand break repair. The absence of Ku and the consequent great shortening of telomeres had no detectable influence either on the rate of VSG switching or on the silencing of the telomeric promoters of the VSG subset that is expressed in the tsetse fly.

Amino Acid Sequence↗
PubMed2002

Constitutive DNase I hypersensitivity of p53-regulated promoters.

The ability of p53 to alter, at the transcriptional level, the gene expression of downstream targets is critical for its role as a tumor suppressor. Most models of p53 activation postulate the stepwise recruitment by p53 of coactivators, histone acetyltransferases, and/or chromatin remodeling factors to a promoter region to facilitate the subsequent access of the general transcriptional machinery required for transcriptional induction. We demonstrate here, however, that the promoter regions for the p53 target genes, p21, 14-3-3sigma, and KARP-1, exist in a constitutively open conformation that is readily accessible to DNase I. This conformation was not altered by DNA damage or by whether p53 was present or absent in the cell. In contrast, p53 response elements, which resided outside the immediate promoter regions, existed within DNase I-resistant chromatin domains. Thus, p53 activation of downstream target genes occurs without p53 inducing chromatin alterations detectable by DNase I accessibility at either the promoter or the response element. As such, these data support models of p53 activation that do not require extensive chromatin alterations to support cognate gene expression.

14-3-3 Proteins↗