Biomedical subjects
Publications and source records attributed to T M Gottlieb.
One of the several biological functions attributed to p53 is the ability to induce apoptotic cell suicide. It has become clear that this apoptotic activity of p53 is central to its role as a tumor suppressor. A summary of current knowledge concerning the mechanisms of p53-mediated apoptosis is presented. The pivotal 'choice' between p53-induced viable growth arrest and apoptosis is discussed.
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DNA-dependent protein kinase is a nuclear serine/threonine kinase whose catalytic properties are expressed only when the enzyme is bound to DNA ends or other discontinuities in the DNA. DNA-PK comprises two components: one mediates binding to DNA and corresponds to the heterodimeric human autoimmune antigen Ku; the other, DNA-PK catalytic subunit (DNA-PKcs), is a polypeptide of approximately 450 kDa. DNA-PK deficiencies are associated with certain mutant rodent cell lines that display defects in DNA double strand break repair and V(D)J recombination. Specifically, hamster xrs-6 cells lack Ku function, whereas murine scid and hamster V3 cells lack functional DNA-PKcs. Furthermore, the phenotypes of xrs-6 and V3 cells can be corrected by the expression of the genes encoding the 80 kDa component of Ku or DNA-PKcs, respectively. These results imply that DNA-PK is an important component of the DNA double strand break repair/recombination apparatus. Possible roles for DNA-PK in these processes are discussed.
Murine cells homozygous for the severe combined immune deficiency mutation (scid) and V3 mutant hamster cells fall into the same complementation group and show similar defects in V(D)J recombination and DNA double-stranded break repair. Here we show that both cell types lack DNA-dependent protein kinase (DNA-PK) activity owing to defects in DNA-PKcs, the catalytic subunit of this enzyme. Furthermore, we demonstrate that yeast artificial chromosomes containing the DNA-PKcs gene complement both the DNA repair and recombination deficiencies of V3 cells, and we conclude that DNA-PKcs is encoded by the XRCC7 gene. As DNA-PK binds to DNA ends and is activated by these structures, our findings provide novel insights into V(D)J recombination and DNA repair processes.
DNA-dependent protein kinase (DNA-PK) comprises a catalytic subunit of approximately 350 kD (p350) and a DNA-binding component termed Ku. Although DNA-PK can phosphorylate many transcription factors, no function for this enzyme in transcription has been reported thus far. Here, we show that DNA-PK strongly represses transcription by RNA polymerase I (Pol I). Transcriptional repression by DNA-PK requires ATP hydrolysis, and DNA-PK must be colocalized on the same DNA molecule as the Pol I transcription machinery. Consistent with DNA-PK requiring DNA ends for activity, transcriptional inhibition only occurs effectively on linearized templates. Mechanistic studies including single-round transcriptions, abortive initiation assays, and factor-independent transcription on a tailed template demonstrate that DNA-PK inhibits initiation (i.e., the formation of the first phosphodiester bonds) but does not affect transcription elongation. Repression of transcription involves phosphorylation of the transcription initiation complex, and rescue experiments reveal that the inactivated factor remains bound to the promoter and thus prevents initiation complex formation. We discuss the possible relevance of these findings in regard to the control of rRNA synthesis in vivo.
DNA-dependent protein kinase (DNA-PK) is a nuclear serine/threonine protein kinase composed of a catalytic subunit called p350 and a DNA binding component termed Ku. Ku consists of two tightly associated polypeptides of approximately 70 kDa and 80 kDa (Ku80). An intriguing feature of DNA-PK is that it binds to DNA ends and other discontinuities in DNA and requires these structures for its activation. This suggests that DNA-PK may function in DNA repair and/or recombination. Consistent with this, Ku DNA binding activity was shown recently to be absent in extracts of hamster xrs-6 cells, which are defective in DNA double-strand (ds) break repair and V(D)J recombination. Furthermore, xrs-6 cells are complemented by expression of the Ku80 cDNA. To date, DNA-PK activity has been demonstrated unequivocally only in extracts of primate cells. Here, we describe an assay that can detect DNA-PK activity in extracts of mouse, hamster, Xenopus, and Drosophila cells. Using this assay, we find that xrs-6 cells completely lack DNA-PK activity. By contrast, xrs-6 derivatives complemented by human chromosome fragments bearing the Ku80 gene have restored both the DNA end binding and kinase activities associated with DNA-PK. Finally, we show that xrs-6 extracts are complemented biochemically by purified Ku. Our findings indicate that the xrs-6 defects are direct consequences of the mutation in Ku80 and implicate DNA-PK in recombination and DNA repair processes.
Inactivation of the TATA-binding protein-containing complex TFIIIB contributes to the mitotic repression of RNA polymerase III transcription, both in frogs and in humans (J. M. Gottesfeld, V. J. Wolf, T. Dang, D. J. Forbes, and P. Hartl, Science 263:81-84, 1994; R. J. White, T. M. Gottlieb, C. S. Downes, and S. P. Jackson, Mol. Cell. Biol. 15:1983-1992, 1995). Using extracts of synchronized proliferating HeLa cells, we show that TFIIIB activity remains low during the early part of G1 phase and increases only gradually as cells approach S phase. As a result, the transcription of all class III genes tested is significantly less active in early G1 than it is in S or G2 phase, both in vitro and in vivo. The increased activity of TFIIIB as cells progress through interphase appears to be due to changes in the TATA-binding protein-associated components of this complex. The data suggest that TFIIIB is an important target for the cell cycle regulation of RNA polymerase III transcription during both mitosis and interphase of actively proliferating HeLa cells.
The mitotic state is associated with a generalized repression of transcription. We show that mitotic repression of RNA polymerase III transcription can be reproduced by using extracts of synchronized HeLa cells. We have used this system to investigate the molecular basis of transcriptional repression during mitosis. We find a specific decrease in the activity of the TATA-binding-protein (TBP)-containing complex TFIIIB. TBP itself is hyperphosphorylated at mitosis, but this does not appear to account for the loss of TFIIIB activity. Instead, one or more TBP-associated components appear to be regulated. The data suggest that changes in the activity of TBP-associated components contribute to the coordinate repression of gene expression that occurs at mitosis.
The radiosensitive mutant xrs-6, derived from Chinese hamster ovary cells, is defective in DNA double-strand break repair and in ability to undergo V(D)J recombination. The human XRCC5 DNA repair gene, which complements this mutant, is shown here through genetic and biochemical evidence to be the 80-kilodalton subunit of the Ku protein. Ku binds to free double-stranded DNA ends and is the DNA-binding component of the DNA-dependent protein kinase. Thus, the Ku protein is involved in DNA repair and in V(D)J recombination, and these results may also indicate a role for the Ku-DNA-dependent protein kinase complex in those same processes.
Failure to remove DNA damage is potentially lethal. Eukaryotic cells have therefore devised highly effective ways of detecting and repairing DNA lesions. Recent evidence indicates that protein kinases play essential roles in recognizing DNA damage and in transducing DNA damage signals to bring about changes in cellular metabolism that facilitate DNA repair.
The DNA-dependent protein kinase (DNA-PK) phosphorylates a number of transcription factors. Here, we show that the DNA-PK modifies c-Jun in vitro and that serine residue 249 (Ser-249) is required for phosphorylation to occur. This residue corresponds to one of three sites of c-Jun that are phosphorylated in vivo and which negatively regulate c-Jun DNA binding in vitro. However, we find that phosphorylation of c-Jun by the DNA-PK does not interfere with DNA binding, indicating that phosphorylation at other sites is required for this effect. Mutagenesis of the phosphorylated region of c-Jun reveals that the primary amino acid sequence recognised by the DNA-PK consists of the sequence Ser-Gln, and that adjacent acidic residues potentiate kinase activity. Furthermore, when this site is placed within the context of a second protein, it confers DNA-PK directed phosphorylation upon that protein. Our findings will facilitate identification of DNA-PK phosphorylation sites in other transcription factors.
The DNA-dependent protein kinase (DNA-PK) phosphorylates Sp1 and several other nuclear proteins. Here, we show that Sp1 and the DNA-PK must be colocalized on the same DNA molecule for efficient phosphorylation to occur. Interestingly, we find that the DNA-PK binds to and is activated by the ends of DNA molecules. Furthermore, we show that the DNA binding properties of the DNA-PK are identical to those of Ku, a well-characterized human autoimmune antigen. We demonstrate that the DNA-PK can be fractionated into two components, one of which is Ku and the other of which is a polypeptide of approximately 350 kd. DNA cross-linking and coimmunoprecipitation studies indicate that the catalytic 350 kd DNA-PK component is directed to DNA by protein-protein interactions with Ku. The implications of the unusual DNA binding mode and multicomponent nature of the DNA-PK are discussed.