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Results for “Ku Autoantigen”

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At least 649 records · Page 36Linked to original sources
PubMed1998

Apoptosis and expression of DNA repair proteins in ischaemic brain injury in man.

We examined the timing of apoptosis and the expression of the DNA repair proteins poly(ADP-ribose) polymerase (PARP) and Ku80 in sections of frontal and temporal lobes from patients who had suffered severe brain ischaemia due to a cardiac arrent. In situ end-labelling (ISEL) was used to detect apoptotic cells, and immunohistochemistry to assess PARP and Ku80. ISEL of scattered neurons and glia was demonstrable predominantly during the first 24 h after ischaemia. PARP and Ku80 immunoreactivity increased markedly after cerebral ischaemia, PARP particularly in the regions of greatest susceptibility to hypoxic injury: the CA1 field of the hippocampus and the depths of neocortical sulci. The up-regulation of PARP is in keeping with experimental observations concerning the key role of this enzyme in mediating ischaemic cell death.

Adult↗
PubMed2003

Expression of Ku80 in cervical cancer correlates with response to radiotherapy and survival.

To reveal the genes relevant for prediction of cervical cancer after radiotherapy, we previously carried out cDNA microarray experiments on primary cervical cancer comparing patients with a complete response (CR) and those with no change (NC). Some of these genes had already been associated with the radiation response, such as x-ray repair cross-complementing 5 (XRCC5), which was found more in radioresistant tumors than in radiosensitive ones. The aim of this study was to confirm the possible roles of XRCC5 mRNA levels by a real-time polymerase chain reaction method in 20 cervical cancers, and Ku80 protein, which is the gene product of XRCC5, using a histopathologic method of formalin-fixed sections of tumor biopsies in determining tumor response to radiotherapy and survival in 89 patients with cervical cancer. The levels of XRCC5 mRNA were 10(4.82) +/- 10(0.2) copies/microg total RNA in tumor tissues in the CR group (mean +/- standard deviation) and 10(4.95) +/- 10(0.32) copies/microg total RNA in those in the NC group. The levels of XRCC5 mRNA were not significantly different between the CR and NC groups. Histopathologic methods revealed 29.2% (26 of 89) of the patients to be Ku80-negative, with Ku80-positive findings in 70.8% (63 of 89). Of the Ku80-negative patients, 19 had CR, 3 had a partial response (PR), and 4 had NC. Of the Ku80-positive patients, 25 had CR, 22 had PR, and 16 had NC. Ku80-negative tumors showed significantly better responses than Ku80-positive ones, comparing CR and PR/NC responses (p = 0.01). In addition, overall survival was significantly better in the Ku80-negative patients as compared with those who were Ku80-positive (p = 0.04). The results of this study suggest that a low expression of Ku80 protein leads to radiosensitivity in cervical cancer and that Ku80 might play a role in treatment outcome.

Aged↗
PubMed1996

Drosophila IRBP/Ku p70 corresponds to the mutagen-sensitive mus309 gene and is involved in P-element excision in vivo.

The P family of transposable elements in Drosophila transpose by a cut-and-paste mechanism involving double-strand gap repair. We report here that a Drosophila mutagen-sensitive mutant, mus3O9, contains a mutation in IRBP (inverted repeat binding protein), the Drosophila homolog of the mammalian Ku p70 gene. We show that the repair of double-strand DNA breaks after P-element excision is severely reduced in mus3O9 mutants using an in vivo assay for P-element transposase activity. In addition, excision products recovered from mus3O9 mutant embryos by use of a plasmid-based P-element mobility assay contain large deletions, suggesting that IRBP is involved in the repair of double-strand DNA breaks. Our findings provide the first demonstration that a mutation in the IRBP gene affects double-strand DNA break repair and suggest that DNA repair functions are conserved between Drosophila and mammals.

Animals↗
PubMed2002

Autophosphorylation of the DNA-dependent protein kinase catalytic subunit is required for rejoining of DNA double-strand breaks.

Nonhomologous end-joining (NHEJ) is the predominant pathway that repairs DNA double-strand breaks (DSBs) in mammalian cells. The DNA-dependent protein kinase (DNA-PK), consisting of Ku and DNA-PK catalytic subunit (DNA-PKcs), is activated by DNA in vitro and is required for NHEJ. We report that DNA-PKcs is autophosphorylated at Thr2609 in vivo in a Ku-dependent manner in response to ionizing radiation. Phosphorylated DNA-PKcs colocalizes with both gamma-H2AX and 53BP1 after DNA damage. Mutation of Thr2609 to Ala leads to radiation sensitivity and impaired DSB rejoining. These findings establish that Ku-dependent phosphorylation of DNA-PKcs at Thr2609 is required for the repair of DSBs by NHEJ.

Amino Acid Sequence↗
PubMed2003

Ku interacts with telomerase RNA to promote telomere addition at native and broken chromosome ends.

Ku is a conserved DNA end-binding protein that plays various roles at different kinds of DNA ends. At telomeres, Ku is part of the structure that protects the chromosome end, whereas at broken DNA ends, Ku promotes DNA repair as part of the nonhomologous end-joining (NHEJ) pathway. Here, we present evidence of a new role for Ku that impacts both telomere-length maintenance and DNA repair in Saccharomyces cerevisiae. We show that Ku binds TLC1, the RNA component of telomerase. We also describe a novel separation-of-function allele of Ku that is specifically defective in TLC1 binding. In this mutant, telomeres are short and the kinetics of telomere addition are slow, but other Ku-dependent activities, such as chromosome end protection and NHEJ, are unaffected. At low frequency, yeast will use telomerase to heal DNA damage by capping the broken chromosome with telomeric DNA sequences. We show that when Ku's ability to bind TLC1 is disrupted, DNA repair via telomere healing is reduced 10- to 100-fold, and the spectrum of sequences that can acquire a telomere changes. Thus, the interaction between Ku and TLC1 RNA enables telomerase to act at both broken and normal chromosome ends.

Alleles↗
PubMed2004

Collaboration of homologous recombination and nonhomologous end-joining factors for the survival and integrity of mice and cells.

Homologous recombination (HR) and nonhomologous end-joining (NHEJ) are mechanistically distinct DNA repair pathways that contribute substantially to double-strand break (DSB) repair in mammalian cells. We have combined mutations in factors from both repair pathways, the HR protein Rad54 and the DNA-end-binding factor Ku80, which has a role in NHEJ. Rad54(-/-)Ku80(-/-) mice were severely compromised in their survival, such that fewer double mutants were born than expected, and only a small proportion of those born reached adulthood. However, double-mutant mice died at lower frequency from tumors than Ku80 single mutant mice, likely as a result of rapid demise at a young age from other causes. When challenged with an exogenous DNA damaging agent, ionizing radiation, double-mutant mice were exquisitely sensitive to low doses. Tissues and cells from double-mutant mice also showed indications of spontaneous DNA damage. Testes from some Rad54(-/-)Ku80(-/-) mice displayed enhanced apoptosis and reduced sperm production, and embryonic fibroblasts from Rad54(-/-)Ku80(-/-) animals accumulated foci of gamma-H2AX, a marker for DSBs. The substantially increased DNA damage response in the double mutants implies a cooperation of the two DSB repair pathways for survival and genomic integrity in the animal.

Aging↗
PubMed1999

Nbs1 potentiates ATP-driven DNA unwinding and endonuclease cleavage by the Mre11/Rad50 complex.

The Nijmegen breakage syndrome gene product (Nbs1) was shown recently to associate in vivo with the Mre11 and Rad50 proteins, which play pivotal roles in eukaryotic DNA double-strand break repair, meiotic recombination, and telomere maintenance. We show in this work that the triple complex of recombinant Nbs1, Mre11, and Rad50 proteins binds cooperatively to DNA and forms a distinct protein-DNA species. The Mre11/Rad50/Nbs1 complex displays several enzymatic activities that are not seen without Nbs1, including partial unwinding of a DNA duplex and efficient cleavage of fully paired hairpins. Unwinding and hairpin cleavage are both increased by the presence of ATP. On nonhairpin DNA ends, ATP controls a switch in endonuclease specificity that allows Mre11/Rad50/Nbs1 to cleave a 3'-protruding strand at a double-/single-strand transition. Mutational analysis demonstrates that Rad50 is responsible for ATP binding by the complex, but the ATP-dependent activities are expressed only with Nbs1 present.

Adenosine Triphosphate↗
PubMed2001

NHEJ regulation by mating type is exercised through a novel protein, Lif2p, essential to the ligase IV pathway.

In the yeast Saccharomyces cerevisiae, DNA double strand break (DSB) repair by nonhomologous end-joining (NHEJ) requires the DNA end-binding heterodimer Yku70p-Yku80p and the ligase Dnl4p associated with its cofactor Lif1p. NHEJ efficiency is down-regulated in MATa/MATalpha cells relative to MATa or MATalpha cells, but the mechanism of this mating type regulation is unknown. Here we report the identification of Lif2p, a S. cerevisiae protein that interacts with Lif1p in a two-hybrid system. Disruption of LIF2 abolishes the capacity of cells to repair DSBs by end-joining to the same extent than lif1 and dnl4 mutants. In MATa/MATalpha cells, Lif2p steady-state level is strongly repressed when other factors involved in NHEJ are unaffected. Increasing the dosage of the Lif2p protein can suppress the NHEJ defect in a/alpha cells. Together, these results indicate that NHEJ regulation by mating type is achieved, at least in part, by a regulation of Lif2p activity.

Antigens, Nuclear↗
PubMed2002

EXO1-dependent single-stranded DNA at telomeres activates subsets of DNA damage and spindle checkpoint pathways in budding yeast yku70Delta mutants.

We have examined the role of checkpoint pathways in responding to a yku70Delta defect in budding yeast. We show that CHK1, MEC1, and RAD9 checkpoint genes are required for efficient cell cycle arrest of yku70Delta mutants cultured at 37 degrees C, whereas RAD17, RAD24, MEC3, DDC1, and DUN1 play insignificant roles. We establish that cell cycle arrest of yku70Delta mutants is associated with increasing levels of single-stranded DNA in subtelomeric Y' regions, and find that the mismatch repair-associated EXO1 gene is required for both ssDNA generation and cell cycle arrest of yku70Delta mutants. In contrast, MRE11 is not required for ssDNA generation. The behavior of yku70Delta exo1Delta double mutants strongly indicates that ssDNA is an important component of the arrest signal in yku70Delta mutants and demonstrates a link between damaged telomeres and mismatch repair-associated exonucleases. This link is confirmed by our demonstration that EXO1 also plays a role in ssDNA generation in cdc13-1 mutants. We have also found that the MAD2 but not the BUB2 spindle checkpoint gene is required for efficient arrest of yku70Delta mutants. Therefore, subsets of both DNA-damage and spindle checkpoint pathways cooperate to regulate cell division of yku70Delta mutants.

Antigens, Nuclear↗
PubMed2000

Ku acts in a unique way at the mammalian telomere to prevent end joining.

Telomeres are specialized DNA/protein structures that act as protective caps to prevent end fusion events and to distinguish the chromosome ends from double-strand breaks. We report that TRF1 and Ku form a complex at the telomere. The Ku and TRF1 complex is a specific high-affinity interaction, as demonstrated by several in vitro methods, and exists in human cells as determined by coimmunoprecipitation experiments. Ku does not bind telomeric DNA directly but localizes to telomeric repeats via its interaction with TRF1. Primary mouse embryonic fibroblasts that are deficient for Ku80 accumulated a large percentage of telomere fusions, establishing that Ku plays a critical role in telomere capping in mammalian cells. We propose that Ku localizes to internal regions of the telomere via a high-affinity interaction with TRF1. Therefore, Ku acts in a unique way at the telomere to prevent end joining.

Animals↗
PubMed2001

Ku DNA end-binding protein modulates homologous repair of double-strand breaks in mammalian cells.

Chromosomal double-strand breaks (DSBs) in mammalian cells are repaired by either homology-directed repair (HDR), using a homologous sequence as a repair template, or nonhomologous end-joining (NHEJ), which often involves sequence alterations at the DSB site. To characterize the interrelationship of these two pathways, we analyzed HDR of a DSB in cells deficient for NHEJ components. We find that the HDR frequency is enhanced in Ku70(-/-), XRCC4(-/-), and DNA-PKcs(-/-) cells, with the increase being particularly striking in Ku70(-/-) cells. Neither sister-chromatid exchange nor gene-targeting frequencies show a dependence on these NHEJ proteins. A Ku-modulated two-ended versus one-ended chromosome break model is presented to explain these results.

Animals↗
PubMed2002

Rap1-Sir4 binding independent of other Sir, yKu, or histone interactions initiates the assembly of telomeric heterochromatin in yeast.

In Saccharomyces cerevisiae, heterochromatin-like regions are found near telomeres and at the silent mating-type loci, where they can repress genes in an epigenetic manner. Several proteins are involved in telomeric heterochromatin structure including Rap1, Sir2, Sir3, Sir4, yKu70 (Hdf1), yKu80 (Hdf2), and the N termini of histones H3 and H4. By recognizing cis-acting DNA-binding sites, Rap1 is believed to recruit Sir and other silencing proteins and determine where heterochromatin forms. The integrity of heterochromatin also requires the binding of Sir proteins to histones that may form a scaffold for Sir protein interactions with chromatin. In this study we describe how the heterochromatin complex may form initially and how it differs from the complex that spreads along the chromosome. We found that close to the telomere end, Sir4 can bind Rap1 independently of Sir2, Sir3, yKu70/yKu80, and the intact H4 N terminus. In contrast, Sir4 binding requires all of the silencing factors further along telomeric heterochromatin. These data indicate that Sir4 binding to Rap1 initiates the sequential association of Sir and other proteins, allowing the subsequent spreading of the heterochromatin proteins along the chromosome.

Antigens, Nuclear↗
PubMed2001

Prokaryotic homologs of the eukaryotic DNA-end-binding protein Ku, novel domains in the Ku protein and prediction of a prokaryotic double-strand break repair system.

Homologs of the eukaryotic DNA-end-binding protein Ku were identified in several bacterial and one archeal genome using iterative database searches with sequence profiles. Identification of prokaryotic Ku homologs allowed the dissection of the Ku protein sequences into three distinct domains, the Ku core that is conserved in eukaryotes and prokaryotes, a derived von Willebrand A domain that is fused to the amino terminus of the core in eukaryotic Ku proteins, and the newly recognized helix-extension-helix (HEH) domain that is fused to the carboxyl terminus of the core in eukaryotes and in one of the Ku homologs from the Actinomycete Streptomyces coelicolor. The version of the HEH domain present in eukaryotic Ku proteins represents the previously described DNA-binding domain called SAP. The Ku homolog from S. coelicolor contains a distinct version of the HEH domain that belongs to a previously unnoticed family of nucleic-acid-binding domains, which also includes HEH domains from the bacterial transcription termination factor Rho, bacterial and eukaryotic lysyl-tRNA synthetases, bacteriophage T4 endonuclease VII, and several uncharacterized proteins. The distribution of the Ku homologs in bacteria coincides with that of the archeal-eukaryotic-type DNA primase and genes for prokaryotic Ku homologs form predicted operons with genes coding for an ATP-dependent DNA ligase and/or archeal-eukaryotic-type DNA primase. Some of these operons additionally encode an uncharacterized protein that may function as nuclease or an Slx1p-like predicted nuclease containing a URI domain. A hypothesis is proposed that the Ku homolog, together with the associated gene products, comprise a previously unrecognized prokaryotic system for repair of double-strand breaks in DNA.

Amino Acid Motifs↗