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Results for “Ku Autoantigen”

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PubMed1997

Ku80 gene expression is Sp1-dependent and sensitive to CpG methylation within a novel cis element.

The Ku70/80 complex, known as Ku, constitutes the DNA end binding component of the DNA-dependent protein kinase (DNA-PK). We have characterized the promoter region of the mouse and human Ku80 genes to delineate transcriptional elements necessary for basal gene expression and proliferation-dependent regulation. Consensus Sp1 recognition elements were identified in both promoters, and were determined to be essential for basal expression. We further identified a near-perfect palindrome of 21 base pairs located immediately 5' to one Sp1 element. This sequence was present once within the mouse Ku80 promoter and seven times, in a head-to-tail tandem array, within the human Ku80 promoter. This sequence possessed homology with a methylation-sensitive promoter element, Enh2, present in the LTR of mouse intractisternal A-particles. Promoter deletion studies and expression analysis of in-vitro methylated reporter gene constructs provided strong evidence that, in vivo, this repeat sequence regulates Ku80 gene expression in cis, through a mechanism involving CpG methylation. Evidence is also presented, suggesting that Ku is directly involved in this regulatory process.

Animals↗
PubMed2003

Evidence implicating Ku antigen as a structural factor in RNA polymerase II-mediated transcription.

Ku antigen is an abundant nuclear protein with multiple functions that depend mainly on Ku's prolific and highly verstatile interactions with DNA. We have shown previously that the direct binding of Ku in vitro to negative regulatory element 1 (NRE1), a transcriptional regulatory element in the long terminal repeat of mouse mammary tumour virus, correlates with the regulation of viral transcription by Ku. In this study, we have sought to explore the interaction of Ku with NRE1 in vivo in yeast one-hybrid experiments. Unexpectedly, we observed that human Ku70 carrying a transcriptional activation domain from the yeast Gal4 protein induced transcription of yeast reporter genes pleiotrophically, independent of NRE1, promoter, reporter gene and chromosomal location. Ku80 with the same activation domain had no effect on transcription when expressed alone, but reconstituted activation when co-expressed with native human Ku70. The requirements for transcriptional activation by Ku-Gal4 activation domain proteins correlated with previous descriptions of the requirements for DNA sequence-independent DNA binding by Ku, but were distinct from determinants for DNA-end binding by a truncated Ku heterodimer determined recently by crystallography. These results suggest a preferential targeting of Ku to transcriptionally active chromatin that indicate a possible function for Ku within the RNA polymerase II holoenzyme.

Animals↗
PubMed2000

Werner syndrome protein: biochemical properties and functional interactions.

Werner syndrome is a premature aging syndrome displaying numerous signs and symptoms found in normal aging. The disease is associated with a mutation in the WRN gene. We have purified the Werner protein (WRN) and studied its biochemical activities and its protein interactions. WRN is a helicase and an exonuclease and also has an associated ATPase activity. WRN interacts physically and functionally with replication protein A (RPA), which stimulates its helicase activity. We have studied the WRN exonuclease activity and found that it can be blocked by certain DNA lesions and not by others. Thus, while WRN does not bind to DNA damage, it may have properties that allow it to sense the presence of damage in DNA. More recently we have found other protein interactions that involve physical and functional interactions with WRN, which could suggest a role for WRN in DNA repair.

Antigens, Nuclear↗
PubMedDate not supplied

The DNA repair protein ku is involved in gp130-mediated signal transduction events in PBMC from young but not from elderly subjects.

Ku, composed of 70kDa (ku 70) and 86kDa (ku 80) proteins, is the DNA-targeting subunit of the DNA-dependent serine/threonine kinase (DNA-PK), which plays a crucial role in DNA double strand break recognition and repair in mammalian cells. We have investigated the effects of an IL-6-type cytokine (K-7/D-6), known to trigger gp130, on the expression and function of the ku protein in cytoplasmic and nuclear extracts of freshly isolated human peripheral blood mononuclear cells (PBMC) from subjects of different ages. DNA-binding of nuclear ku was found to be increased by cytokine treatment of cells from young donors but only to a negligible extent from elderly subjects. This cytokine effect was correlated with a greater amount of phosphorylated ku 80, rather than increased expression of ku 70 and ku 80 proteins. DNA-binding activity of cytoplasmic ku was hardly discernible, as compared to nuclear ku, in both young and elderly subjects and was unaffected by the cytokine treatment regardless of age. Regarding the mechanisms whereby ku and gp130 signaling are coupled in PBMC, results from co-immunoprecipitation experiments have shown that ku in the cytoplasm of PBMC from young, but not from elderly subjects, is associated with Tyk-2, a kinase involved in signal transduction events after gp130 triggering by IL-6-type cytokines. This association was independent of PHA stimulation. Moreover, the present results indicate that after gp130 signaling both Tyk-2 and ku 80 are phosphorylated, suggesting their activation by K-7/D-6.

Adult↗
PubMed1999

Effect of age on DNA binding of the ku protein in irradiated human peripheral blood mononuclear cells (PBMC).

DNA binding of the ku protein was investigated in peripheral blood mononuclear cells (PBMC) from 24 subjects of different ages (20-89 years old) displaying age-related changes in DNA repair, mitotic responsiveness, and cytokine production. Ku is an heterodimeric protein composed of two subunits of 70 and 80 kDa, which is involved in the earliest steps of DNA damage recognition. DNA binding of ku 70/80 was found unchanged in normal PBMC from aging subjects but progressively declined in x-ray-irradiated PBMC from young to adult, and elderly subjects. This finding was concomitant with the age-related fall of DNA repair in the whole population.

Adult↗
PubMed2000

Enhanced sensitivity and long-term G2 arrest in hydrogen peroxide-treated Ku80-null cells are unrelated to DNA repair defects.

While the Ku complex, comprised of Ku70 and Ku80, is primarily involved in the repair of DNA double-strand breaks, it is also believed to participate in additional cellular processes. Here, treatment of embryo fibroblasts (MEFs) derived from either wild-type or Ku80-null (Ku80(-/-)) mice with various stress agents revealed that hydrogen peroxide (H(2)O(2)) was markedly more cytotoxic for Ku80(-/-) MEFs and led to their long-term accumulation in the G2 phase. This differential response was not due to differences in DNA repair, since H(2)O(2)-triggered DNA damage was repaired with comparable efficiency in both Wt and Ku80(-/-) MEFs, but was associated with differences in the expression of important cell cycle regulatory genes. Our results support the notion that Ku80-mediated cytoprotection and G2-progression are not only dependent on the cell's DNA repair but also may reflect Ku80's influence on additional cellular processes such as gene expression.

Animals↗
PubMed2000

Human colon carcinoma cell-line HCT116 transfected by antisense cDNA as a tool to study the Ku86 involvement in cell proliferation.

In this work, we used colon carcinoma cell-line HCT116 to study the involvement of the 86-kDa subunit (Ku86) of DNA-protein kinase (DNA-PK) in human tumoural cell proliferation. We transfected these cells with a 639-bp cDNA encoding a Ku86 portion inserted into pcDNA3.1 vector in the antisense orientation. After selection by neomycin, we obtained more than 300 resistant colonies. In the Y'A5 colony that we chose as total population, we showed by PCR and RT-PCR that pcDNA3/Ku86 antisense was integrated in genomic DNA and that transcript was present. After cloning, we selected two clones, A20 and A23, which contained significatively reduced level of Ku86 protein. These two clones displayed a reduced DNA-PK activity from 44% to 71% and a slower growth than control cells. These results suggest that the HCT116 cell-line is a useful tool to investigate the role of Ku86 in the regulation of human tumoural cell growth.

Antigens, Nuclear↗
PubMed2001

Direct transfer of Ku between DNA molecules with nonhomologous ends.

The Ku protein is an essential protein for DNA double-strand-break repair by the pathway of nonhomologous DNA end-joining (NHEJ). A previous study showed that Ku bound to one DNA molecule could transfer directly to another DNA molecule without being released into the solution first. Direct transfer requires the two DNA molecules having homologous cohesive ends with a minimum of four complementary bases. Results of this study reveal that direct transfer activity of Ku is regulated by NaCl and MgCl2. Increasing either one of the two cations can decrease the required amount of the other. However, the DNA end-binding activity of Ku is not affected by changing the concentration of the cations, indicating that the two activities are regulated independently. Most importantly, the results also show that Ku can transfer directly from one DNA molecule to another one with nonhomologous ends under the condition of 200 mM NaCl and 5mM MgCl2. The ability of direct transfer between DNAs with nonhomologous ends suggests that Ku can align or juxtapose two DNA ends during NHEJ.

Antigens, Nuclear↗
PubMed1997

Interaction between DNA-dependent protein kinase and a novel protein, KIP.

DNA-dependent protein kinase (DNA-PKcs) is the only eukaryotic kinase activated by DNA ends. Mutation of DNA-PKcs results in murine severe combined immune deficiency in mice and radiation sensitivity. Both the immune and the radiation defects are due to a failure in double-strand break repair. Biochemical studies indicate that DNA-PKcs kinase activity is stimulated by the presence of the DNA end binding protein. Ku. Autophosphorylation of DNA-PKcs results in its inactivation. Based on these studies, DNA-PKcs is presumed to play a direct and important role in the repair of double-strand breaks, but the details of its role are quite unclear. We have done two-hybrid analysis of this entire protein to identify other proteins with which it interacts. Thus far, extensive analysis has only revealed one strong interaction that satisfies both high genetic and biochemical stringency. The interaction is with a novel human protein that has 26% amino acid identity with the phosphatase component, calcineurin B. We discuss the interaction of DNA-PKcs with this novel calcium-binding protein family member in the context of possible kinase-phosphatase regulation of DNA end joining.

Adult↗
PubMed1999

Ku, a DNA repair protein with multiple cellular functions?

The Ku protein binds to DNA ends and other types of discontinuity in double-stranded DNA. It is a tightly associated heterodimer of approximately 70 kDa and approximately 80 kDa subunits that together with the approximately 470 kDa catalytic subunit, DNA-PKcs, form the DNA-dependent protein kinase. This enzyme is involved in repairing DNA double-strand breaks (DSBs) caused, for example, by physiological oxidation reactions, V(D)J recombination, ionizing radiation and certain chemotherapeutic drugs. The Ku-dependent repair process, called illegitimate recombination or nonhomologous end joining (NHEJ), appears to be the main DNA DSB repair mechanism in mammalian cells. Ku itself is probably involved in stabilizing broken DNA ends, bringing them together and preparing them for ligation. Ku also recruits DNA-PKcs to the DSB, activating its kinase function. Targeted disruption of the genes encoding Ku70 and Ku80 has identified significant differences between Ku-deficient mice and DNA-PKcs-deficient mice. Although all three gene products are clearly involved in repairing ionizing radiation-induced damage and in V(D)J recombination, Ku-knockout mice are small, and their cells fail to proliferate in culture and show signs of premature senescence. Recent findings have implicated yeast Ku in telomeric structure in addition to NHEJ. Some of the phenotypes of the Ku-knockout mice may indicate a similar role for Ku at mammalian telomeres.

Animals↗
PubMed1999

Senescent human fibroblasts have elevated Ku86 proteolytic cleavage activity.

A proteolytic activity capable of cleaving the Ku86 subunit of Ku protein to two polypeptides, with molecular masses of 69 and 17 kDa in vitro, is present in a human diploid fibroblast (HDF) cell line. The activity is elevated in late-passaged and senescent cells, and the cleaved 69-kDa product seems able to form complex with Ku70 to bind DNA ends. However, further studies indicate that cleavage of Ku86 could be inhibited by including leupeptin in the extraction buffer, and no 69 kDa variant was evident in the cell. In fact, the levels of Ku86, Ku70 and DNA-end binding activity of Ku remained unchanged during replicative senescence. Thus, this study reveals an intriguing protease in HDFs, and also indicates that inconsistent results of Ku86 expression will be obtained if the protease activity is not completely inhibited.

Antigens, Nuclear↗
PubMed2002

Mechanisms of chromosome-end protection.

Telomeres must protect chromosome ends from being recognized and processed as double-strand breaks. Identification of the factors involved in end protection, and the mechanisms by which they "cap" chromosome termini, is crucial in understanding how the cell distinguishes between a double-strand break and a normal telomere end. Recent work has characterized the similarities and potential differences between the pathways utilized by multiple organisms in maintaining telomere ends. One unifying concept that has clearly emerged is that chromosome-end protection is necessary in maintaining genetic stability and preventing oncogenesis.

Antigens, Nuclear↗
PubMed1997

Tying loose ends: roles of Ku and DNA-dependent protein kinase in the repair of double-strand breaks.

A convergence of information from biochemistry, yeast and mammalian genetics, immunology, and radiation biology has permitted identification of some of the protein participants - Ku, DNA-PK, XRCC4 - and the reaction intermediates in DNA end joining, suggesting how broken chromosomal ends may be recognized and repaired in eukaryotic cells. Some components may be defective in inherited disorders.

Animals↗
PubMed1999

Telomere maintenance mechanisms and cellular immortalization.

Immortal cell populations are able to proliferate indefinitely. Immortalization is associated with activation of processes that compensate for the telomeric shortening that accompanies cell division in normal somatic cells. In many immortal cell lines, telomere maintenance is provided by the action of the ribonucleoprotein enzyme complex, telomerase. Some immortal cell lines have undetectable or very low levels of telomerase activity and there is evidence that these cells maintain their telomeres by an alternative mechanism.

Animals↗
PubMed2002

DNA double-strand break repair from head to tail.

DNA double-strand break repair is a complex process that requires multiple enzymatic and structural activities to rejoin or repair the broken DNA ends using one of several repair pathways. These enzymatic and structural activities include end detection, end processing and alignment of DNA ends. Recent structural and functional studies of the DNA double-strand break repair factors Mre11/Rad50, Ku70/80 and Xrcc4 show how these enzymes combine and assemble both enzymatic and structural activities in DNA double-strand break repair.

Adenosine Triphosphate↗
PubMed1994

Recombination and repair. Ku starts at the end.

Genetic evidence suggests that the Ku DNA-end-binding protein complex is central to the recombination-based repair of double-strand breaks that protects DNA from radiation and underlies antibody gene rearrangement.

Animals↗
PubMed2000

Lif1p targets the DNA ligase Lig4p to sites of DNA double-strand breaks.

DNA ligases catalyse the joining of DNA single- and double-strand breaks. Saccharomyces cerevisiae Cdc9p is a homologue of mammalian DNA ligase I and is required for DNA replication, recombination and single-strand break repair. The other yeast ligase, Lig4p/Dnl4p, is a homologue of mammalian DNA ligase IV, and functions in the non-homologous end-joining (NHEJ) pathway of DNA double-strand break repair [1] [2] [3] [4]. Lig4p interacts with Lif1p, the yeast homologue of the human ligase IV-associated protein, XRCC4 [5]. This interaction takes place through the carboxy-terminal domain of Lig4p and is required for Lig4p stability. We show that the carboxy-terminal interaction region of Lig4p is necessary for NHEJ but, when fused to Cdc9p, is insufficient to confer NHEJ function to Cdc9p. Also, Lif1p stimulates the in vitro catalytic activity of Lig4p in adenylation and DNA ligation. Nevertheless, Lig4p is inactive in NHEJ in the absence of Lif1p in vivo, even when Lig4p is stably expressed. We show that Lif1p binds DNA in vitro and, through in vivo cross-linking and chromatin immuno precipitation assays, demonstrate that it targets Lig4p to chromosomal DNA double-strand breaks. Furthermore, this targeting requires another key NHEJ protein, Ku.

Antigens, Nuclear↗