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D T Weaver

Publications and source records attributed to D T Weaver.

At least 19 recordsLinked to original sources
PubMed2001

Rescue of a telomere length defect of Nijmegen breakage syndrome cells requires NBS and telomerase catalytic subunit.

Nijmegen breakage syndrome (NBS) is a rare human disease displaying chromosome instability, radiosensitivity, cancer predisposition, immunodeficiency, and other defects [1, 2]. NBS is complexed with MRE11 and RAD50 in a DNA repair complex [3-5] and is localized to telomere ends in association with TRF proteins [6, 7]. We show that blood cells from NBS patients have shortened telomere DNA ends. Likewise, cultured NBS fibroblasts that exhibit a premature growth cessation were observed with correspondingly shortened telomeres. Introduction of the catalytic subunit of telomerase, TERT, was alone sufficient to increase the proliferative capacity of NBS fibroblasts. However, NBS, but not TERT, restores the capacity of NBS cells to survive gamma irradiation damage. Strikingly, NBS promotes telomere elongation in conjunction with TERT in NBS fibroblasts. These results suggest that NBS is a required accessory protein for telomere extension. Since NBS patients have shortened telomeres, these defects may contribute to the chromosome instability and disease associated with NBS patients.

Catalytic Domain↗
PubMed2000

ATM phosphorylation of Nijmegen breakage syndrome protein is required in a DNA damage response.

Nijmegen breakage syndrome (NBS) is characterized by extreme radiation sensitivity, chromosomal instability and cancer. The phenotypes are similar to those of ataxia telangiectasia mutated (ATM) disease, where there is a deficiency in a protein kinase that is activated by DNA damage, indicating that the Nbs and Atm proteins may participate in common pathways. Here we report that Nbs is specifically phosphorylated in response to gamma-radiation, ultraviolet light and exposure to hydroxyurea. Phosphorylation of Nbs mediated by gamma-radiation, but not that induced by hydroxyurea or ultraviolet light, was markedly reduced in ATM cells. In vivo, Nbs was phosphorylated on many serine residues, of which S343, S397 and S615 were phosphorylated by Atm in vitro. At least two of these sites were underphosphorylated in ATM cells. Inactivation of these serines by mutation partially abrogated Atm-dependent phosphorylation. Reconstituting NBS cells with a mutant form of Nbs that cannot be phosphorylated at selected, ATM-dependent serine residues led to a specific reduction in clonogenic survival after gamma-radiation. Thus, phosphorylation of Nbs by Atm is critical for certain responses of human cells to DNA damage.

Ataxia Telangiectasia↗
PubMed2000

Amniotic tissue in complete hydatidiform moles can be androgenetic.

The purpose of this study was to determine whether amniotic tissue found associated with cases of complete hydatidiform mole (CM) was genetically identical to the CM, and therefore part of the molar pregnancy, or genetically dissimilar to the CM, suggesting derivation from a twin pregnancy. DNA was prepared from formalin-fixed, paraffin-embedded blocks of tissue containing both CM and amnion. Maternal DNA was prepared from decidual tissue in the same blocks, or from a maternal blood sample. Fluorescent microsatellite genotyping was carried out to determine the origin of both the CM and the amniotic tissue. In one of six cases examined, the amniotic tissue was genetically different from the CM and was therefore likely to be derived from a twin pregnancy. In the five remaining cases, the amniotic tissue was genetically identical to the CM and was likely to be derived from the same conceptus. It is concluded that androgenetic CM can support the development of amniotic tissue and that some early embryonic development may occur in CM. The presence of amnion, or other fetal tissues, associated with molar tissue should not therefore always be considered indicative of a diagnosis of partial mole (PM).

Alleles↗
PubMed1998

Enforced CDK4 expression in a hematopoietic cell line confers resistance to the G1 arrest induced by ionizing radiation.

In hematopoietic cells, gamma-irradiation causes a p53-dependent transient G1 phase cell cycle arrest. Various extracellular growth inhibitory signals elicit G1 arrest by targeting CDK4. Here we show that in a myeloid cell line, 32D cl 3, enforced expression of CDK4, but not cyclins D2 nor D3, overrides the gamma-irradiation-induced G1 arrest. CDK4 does not confer resistance to the radiation-induced G2 block observed in parental cells. Ectopic expression of CDK4 overcomes the ionizing radiation-induced inhibition of CDK4 and CDK2 kinase activity. The levels of CDK4 protein do not change after exposure to ionizing radiation in either parental cells or those overexpressing CDK4. Ionizing radiation induces the expression of both p53 and p21, and in cells constitutively synthesizing exogenous CDK4, the return of p53 protein levels to baseline is prolonged. Increased levels of p21 are found associated with CDK4, and not CDK2, in the lines overexpressing CDK4, compared to the parental line, after exposure to ionizing radiation. Enforced expression of CDK4 may therefore overcome a gamma-irradiation-induced G1 arrest through the titration of the CDK inhibitor p21 allowing both CDK4 and CDK2 to remain active.

Animals↗
PubMed1998

Telomeres: moonlighting by DNA repair proteins.

Chromosome ends, or telomeres, are dynamic DNA structures maintained by a multisubunit telomerase and other proteins. New evidence indicates that proteins previously implicated in the repair of DNA doublestrand breaks also play an important role in the control of telomere organization and length.

Animals↗
PubMed1998

A targeted DNA-PKcs-null mutation reveals DNA-PK-independent functions for KU in V(D)J recombination.

The DNA-dependent protein kinase (DNA-PK) consists of Ku70, Ku80, and a large catalytic subunit, DNA-PKcs. Targeted inactivation of the Ku70 or Ku80 genes results in elevated ionizing radiation (IR) sensitivity and inability to perform both V(D)J coding-end and signal (RS)-end joining in cells, with severe growth retardation plus immunodeficiency in mice. In contrast, we now demonstrate that DNA-PKcs-null mice generated by gene-targeted mutation, while also severely immunodeficient, exhibit no growth retardation. Furthermore, DNA-PKcs-null cells are blocked for V(D)J coding-end joining, but retain normal RS-end joining. Finally, while DNA-PK-null fibroblasts exhibited increased IR sensitivity, DNA-PKcs-deficient ES cells did not. We conclude that Ku70 and Ku80 may have functions in V(D)J recombination and DNA repair that are independent of DNA-PKcs.

Animals↗
PubMed1998

Differential etoposide sensitivity of cells deficient in the Ku and DNA-PKcs components of the DNA-dependent protein kinase.

Etoposides block cell division by interfering with the action of topoisomerase II, leaving enzyme-DNA double-strand breaks. We found that certain components of the trimeric DNA-dependent protein kinase influence cell survival following etoposide damage. Interestingly, either Ku70- or Ku80-deficient cell lines, but not mutant cell lines of the DNA-PK catalytic sub-unit (DNA-PKcs), were found to be hypersensitive to the effects of etoposide VP16. Ku70- and Ku80-deficient cells can be complemented to an etoposide resistant phenotype by introducing wildtype Ku70 or Ku80 cDNAs. Mutational analysis of introduced Ku70 cDNAs into murine embryonic stem cells deleted for Ku70 (-/-) showed that mutants where heterodimerization and DNA binding functions of Ku were disrupted, also blocked the restoration of etoposide resistance. In contrast with the differential etoposide sensitivity of DNA-PK mutants, both Ku- and DNA-PKcs-deficient cell lines showed G2 ionizing radiation-induced delays, a cell cycle phase where topoisomerase II function is critical. Thus, the topoisomerase II cleaved complexes may be an example of DNA lesions requiring the Ku heterodimer, but not DNA-PK for DNA repair.

Animals↗
PubMed1997

Double-strand break repair by Ku70 requires heterodimerization with Ku80 and DNA binding functions.

Heterodimers of the 70 and 80 kDa Ku autoantigens (Ku70 and Ku80) activate the DNA-dependent protein kinase (DNA-PK). Mutations in any of the three subunits of this protein kinase (Ku70, Ku80 and DNA-PKcs) lead to sensitivity to ionizing radiation (IR) and to DNA double-strand breaks, and V(D)J recombination product formation defects. Here we show that the IR repair, DNA end binding and DNA-PK defects in Ku70-/- embryonic stem cells can be counteracted by introducing epitope-tagged wild-type Ku70 cDNA. Truncations and chimeras of Ku70 were used to identify the regions necessary for DNA end binding and IR repair. Site-specific mutational analysis revealed a core region of Ku70 responsible for DNA end binding and heterodimerization. The propensity for Ku70 to associate with Ku80 and to bind DNA correlates with the ability to activate DNA-PK, although two mutants showed that the roles of Ku70 in DNA-PK activation and IR repair are separate. Mutation of DNA-PK autophosphorylation sites and other structural motifs in Ku70 showed that these sites are not necessary for IR repair in vivo. These studies reveal Ku70 features required for double-strand break repair.

Amino Acid Sequence↗
PubMed1997

Binding of Ku and c-Abl at the kinase homology region of DNA-dependent protein kinase catalytic subunit.

The DNA-dependent protein kinase (DNA-PK) controls the repair of double-stranded DNA breaks in mammalian cells. The protein kinase subunit of DNA-PK (DNA-PKcs) is targeted to DNA breaks by association with the Ku DNA-binding heterodimer. Here we show that a Ku association site is present at the carboxyl terminus of DNA-PKcs (amino acids 3002-3850) near the protein kinase domain. Correspondingly, the nuclear c-Abl tyrosine kinase that associates with DNA-PK also binds to the kinase homology domain. The c-Abl SH3 domain binds to amino acids 3414-3850 of DNA-PKcs. c-Abl phosphorylates C-terminal fragments of DNA-PKcs, particularly amino acids 3414-3850. c-Abl phosphorylation of DNA-PKcs disassociates the DNA-PKcs.Ku complex. Thus, Ku and c-Abl provide opposing functions with regard to DNA-PK activity.

Animals↗
PubMed1997

Conditional gene targeted deletion by Cre recombinase demonstrates the requirement for the double-strand break repair Mre11 protein in murine embryonic stem cells.

Repair of DNA damage resulting in double-strand breaks (DSBs) is controlled by gene products executing homologous recombination or end-joining pathways. The MRE11 gene has previously been implicated in DSB repair in the yeast Saccharomyces cerevisiae . Here we have developed a methodology to study the roles of the murine Mre11 homolog in pluripotent embryonic stem cells. Using a gene targeting approach, a triple LoxP site cassette was inserted into a region of MRE11 genomic DNA flanking conserved phosphodiesterase motifs. The addition of Cre recombinase activity promotes deletions of three types that can be scored. We find that deletion at phosphodiesterase motif III encoded in the N-terminus of Mre11 is acheived in the presence of a wild-type MRE11 allele. However, when the wild-type MRE11 allele is inactivated by gene targeted insertion of a neo marker, only Cre recombination events that allow expression of wild-type Mre11 protein are observed. Therefore, Mre11 is required for normal cell proliferation. This methodology introduces a means to study important regions of essential genes in cell culture models.

Amino Acid Sequence↗
PubMed1997

Ku70-deficient embryonic stem cells have increased ionizing radiosensitivity, defective DNA end-binding activity, and inability to support V(D)J recombination.

V(D)J recombination requires both lymphoid-specific and generally expressed enzymatic activities. All three known generally expressed activities involved in V(D)J recombination are also involved in DNA double-strand break repair (DSBR). Two of these are components of the DNA-dependent protein kinase (DNA-PK) and include Ku80 and DNA-PK catalytic subunit (DNA-PKcs); the third, XRCC4, is a protein of unknown function. The Ku70 protein is an additional component of DNA-PK; Ku70 forms a heterodimer with Ku80 to generate the DNA end-binding component of the enzyme. To test putative functions for Ku70, we have used gene-targeted mutation to generate a murine embryonic stem cell line which lacks Ku70 expression. We find that the Ku70(-/-) cells produce no detectable Ku70 and very little Ku80, suggesting a direct interrelationship between their levels. Correspondingly, these cells lack the nonspecific DNA end-binding activity associated with Ku. Significantly, the Ku70(-/-) embryonic stem cells have markedly increased sensitivity to gamma-irradiation relative to Ku70(+/-) or wild-type embryonic stem cells. Furthermore, the Ku70(-/-) cells lack the ability to effectively rejoin signal and coding ends liberated in transiently introduced V(D)J recombination substrates by enforced RAG-1 and RAG-2 expression. We conclude that the Ku70 gene product is involved in DSBR and V(D)J recombination and confirm that the Ku70 gene can be classified as a member of the x-ray cross-complementation group 6 (XRCC6). Potential differences between the Ku70(-/-) and Ku80(-/-) V(D)J recombination defects are discussed.

Animals↗
PubMed1997

Functions of the DNA dependent protein kinase.

The DNA dependent protein kinase (DNA-PK) is a trimeric nuclear complex consisting of a large protein kinase and the Ku heterodimer that regulates kinase activity by its association with DNA. Recent findings have shown structural similarities between DNA-PK and a family of lipid and putative protein kinases (PIK family). DNA-PK is one of the PIK members known to be a protein kinase with clearly identified effector subunits. A broad range of observations link DNA-PK to dual roles in double strand DNA break (DSB) repair and transcription. Unlike its most closely related PIKs, DNA-PK is not required for activating cell cycle regulated DNA damage signalling mechanisms. Instead, the phenotypes and biochemical properties of DNA-PK are most consistent with functions in DSB repair and joining steps in recombination mechanisms. DNA-PK is associated with RNA polymerase II and RNA polymerase I transcription complexes, where it most frequently has a negative regulatory role.

Animals↗
PubMed1996

Chromosomal double-strand break repair in Ku80-deficient cells.

The x-ray sensitive hamster cell line xrs-6 is deficient in DNA double-strand break (DSB) repair and exhibits impaired V(D)J recombination. The molecular defect in this line is in the 80-kDa subunit of the Ku autoantigen, a protein that binds to DNA ends and recruits the DNA-dependent protein kinase to DNA. Using an I-SceI endonuclease expression system, chromosomal DSB repair was examined in xrs-6 and parental CHO-K1 cell lines. A DSB in chromosomal DNA increased the yield of recombinants several thousand-fold above background in both the xrs-6 and CHO-K1 cells, with recombinational repair of DSBs occurring in as many as 1 of 100 cells electroporated with the endonuclease expression vector. Thus, recombinational repair of chromosomal DSBs can occur at substantial levels in mammalian cells and it is not grossly affected in our assay by a deficiency of the Ku autoantigen. Rejoining of broken chromosome ends (end-joining) near the site of the DSB was also examined. In contrast to recombinational repair, end-joining was found to be severely impaired in the xrs-6 cells. Thus, the Ku protein appears to play a critical role in only one of the chromosomal DSB repair pathways.

Animals↗
PubMed1996

Mutations in two Ku homologs define a DNA end-joining repair pathway in Saccharomyces cerevisiae.

DNA double-strand break (DSB) repair in mammalian cells is dependent on the Ku DNA binding protein complex. However, the mechanism of Ku-mediated repair is not understood. We discovered a Saccharomyces cerevisiae gene (KU80) that is structurally similar to the 80-kDa mammalian Ku subunit. Ku8O associates with the product of the HDF1 gene, forming the major DNA end-binding complex of yeast cells. DNA end binding was absent in ku80delta, hdf1delta, or ku80delta hdf1delta strains. Antisera specific for epitope tags on Ku80 and Hdf1 were used in supershift and immunodepletion experiments to show that both proteins are directly involved in DNA end binding. In vivo, the efficiency of two DNA end-joining processes were reduced >10-fold in ku8Odelta, hdfldelta, or ku80delta hdf1delta strains: repair of linear plasmid DNA and repair of an HO endonuclease-induced chromosomal DSB. These DNA-joining defects correlated with DNA damage sensitivity, because ku80delta and hdf1delta strains were also sensitive to methylmethane sulfonate (MMS). Ku-dependent repair is distinct from homologous recombination, because deletion of KU80 and HDF1 increased the MMS sensitivity of rad52delta. Interestingly, rad5Odelta, also shown here to be defective in end joining, was epistatic with Ku mutations for MMS repair and end joining. Therefore, Ku and Rad50 participate in an end-joining pathway that is distinct from homologous recombinational repair. Yeast DNA end joining is functionally analogous to DSB repair and V(D)J recombination in mammalian cells.

Amino Acid Sequence↗