Because only a small fraction of asbestos-exposed individuals develop malignant mesothelioma, and because mesothelioma clustering is observed in some families, we searched for genetic predisposing ...factors. We discovered germline mutations in the gene encoding BRCA1 associated protein-1 (BAP1) in two families with a high incidence of mesothelioma, and we observed somatic alterations affecting BAP1 in familial mesotheliomas, indicating biallelic inactivation. In addition to mesothelioma, some BAP1 mutation carriers developed uveal melanoma. We also found germline BAP1 mutations in 2 of 26 sporadic mesotheliomas; both individuals with mutant BAP1 were previously diagnosed with uveal melanoma. We also observed somatic truncating BAP1 mutations and aberrant BAP1 expression in sporadic mesotheliomas without germline mutations. These results identify a BAP1-related cancer syndrome that is characterized by mesothelioma and uveal melanoma. We hypothesize that other cancers may also be involved and that mesothelioma predominates upon asbestos exposure. These findings will help to identify individuals at high risk of mesothelioma who could be targeted for early intervention.
A Novel DNA Damage Response Zhang, Sufang; Zhou, Yajing; Trusa, Sandra ...
The Journal of biological chemistry,
05/2007, Letnik:
282, Številka:
21
Journal Article
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Mammalian DNA polymerase (Pol) δ is essential for DNA replication. It consists of four subunits, p125, p50, p68, and p12. We report the discovery that the p12 subunit is rapidly degraded in cultured ...human cells by DNA damage or replication stress brought about by treatments with UV, methyl methanesulfonate, hydroxyurea, and aphidicolin. The degradation of p12 is due to an accelerated rate of proteolysis that is inhibited by the proteasome inhibitors, MG132 and lactacystin. UV treatment converts Pol δ in vivo to the three-subunit form lacking p12. This was demonstrated by its isolation using immunoaffinity chromatography. The three-subunit enzyme retains activity on poly(dA)/oligo(dT) templates but is impaired in its ability to extend singly primed M13 templates, clearly indicating that its in vivo functions are likely to be compromised. This transformation of Pol δ by modification of its quaternary structure is reversible in vitro by the addition of the p12 subunit and could represent a novel in vivo mechanism for the modulation of Pol δ function. UV and hydroxyurea-triggered p12 degradation is blocked in ATR–/– cells but not in ATM–/– cells, thereby demonstrating that p12 degradation is regulated by ATR, the apical kinase that regulates the damage response in S-phase. These findings reveal a novel addition to the cellular repertoire of DNA damage responses that also impacts our understanding of the role of Pol δ in both DNA replication and DNA repair.
Mammalian DNA polymerase (Pol) δ is essential for DNA replication. It consists of four subunits, p125, p50, p68, and p12. We report the discovery that the p12 subunit is rapidly degraded in cultured ...human cells by DNA damage or replication stress brought about by treatments with UV, methyl methanesulfonate, hydroxyurea, and aphidicolin. The degradation of p12 is due to an accelerated rate of proteolysis that is inhibited by the proteasome inhibitors, MG132 and lactacystin. UV treatment converts Pol δ in vivo to the three-subunit form lacking p12. This was demonstrated by its isolation using immunoaffinity chromatography. The three-subunit enzyme retains activity on poly(dA)/oligo(dT) templates but is impaired in its ability to extend singly primed M13 templates, clearly indicating that its in vivo functions are likely to be compromised. This transformation of Pol δ by modification of its quaternary structure is reversible in vitro by the addition of the p12 subunit and could represent a novel in vivo mechanism for the modulation of Pol δ function. UV and hydroxyurea-triggered p12 degradation is blocked in ATR⁻/⁻ cells but not in ATM⁻/⁻ cells, thereby demonstrating that p12 degradation is regulated by ATR, the apical kinase that regulates the damage response in S-phase. These findings reveal a novel addition to the cellular repertoire of DNA damage responses that also impacts our understanding of the role of Pol δ in both DNA replication and DNA repair.
Mammalian DNA polymerase δ (pol δ), a key enzyme of chromosomal DNA replication, consists of four subunits as follows: the catalytic subunit; p125, which is tightly associated with the p50 subunit; ...p68, a proliferating cell nuclear antigen (PCNA)-binding protein; and a fourth subunit, p12. In this study, the functional roles of the p12 subunit of pol δ were studied. The inter-subunit interactions of the p12 subunit were determined by yeast two-hybrid assays and by pulldown assays. These assays revealed that p12 interacts with p125 as well as p50. This dual interaction of p12 suggests that it may serve to stabilize the p125-p50 interaction. p12 was shown to be a novel PCNA-binding protein. This was confirmed by identification of a PCNA-binding motif at its N terminus by binding assays and by site-directed mutagenesis. The activities and reaction products of recombinant pol δ containing a p12 mutant defective in PCNA binding, as well as purified recombinant pol δ and its subassemblies, were analyzed. Our results indicate that p12 contributes to PCNA-dependent pol δ activity, i.e. the p12-PCNA interaction is functional. Our data indicate that both p12 and p68 are required for optimal pol δ activity. This supports the hypothesis that the interaction between pol δ and PCNA is a divalent one that involves p12 and p68. We propose a model in which pol δ interacts with PCNA via at least two of its subunits, and one in which p12 could play a role in stabilizing the overall pol δ-PCNA complex as well as pol δ itself.
We previously demonstrated that angiotensin II (Ang II) stimulates an increase in nitric oxide synthase (NOS) mRNA levels, eNOS protein expression and NO production via the type 2 (AT
2) receptor, ...whereas signaling via the type 1 (AT
1) receptor negatively regulates NO production in bovine pulmonary artery endothelial cells (BPAECs). In the present study, we investigated the components of the AT
1 receptor-linked signaling pathway(s) that are involved in the downregulation of eNOS protein expression in BPAECs. Treatment of BPAECs with either AT
1 receptor antagonists or an anti-AT
1 receptor antibody induced eNOS protein expression. Furthermore, intracellular delivery of GP-Antagonist-2A, an inhibitor of Gαq proteins, and treatment of BPAECs with U73122, a phosphatidylinositol-phospholipase C (PLC)-specific inhibitor, enhanced eNOS protein expression. Treatment of BPAECs with the cell-permeable calcium chelator, BAPTA/AM, increased eNOS protein expression at 8 h, while increasing intracellular calcium with either thapsigargin or A23187 prevented Ang II-induced eNOS protein expression. Phorbol myristate acetate (PMA), a protein kinase C (PKC) activator, completely prevented Ang II-stimulated eNOS protein expression at 8 h, whereas depletion of PKC by long-term treatment with PMA, induced eNOS protein expression. Treatment of BPAECs with a PKCα-specific inhibitor or transfection of BPAECs with an anti-PKCα neutralizing antibody stimulated eNOS protein expression. Conversely, rottlerin, a PKCδ specific isoform inhibitor had no effect on basal or Ang II-stimulated eNOS protein expression. Moreover, treatment of BPAECs with U73122, BAPTA/AM and PKCα-specific inhibitors increased NO production at 8 h. In conclusion, Ang II downregulates eNOS protein expression via an AT
1 receptor-linked pathway involving Gαq/PLC/calcium/PKCα signaling pathway in BPAECs.
Mammalian DNA polymerase δ (pol δ), a key enzyme of chromosomal DNA replication, consists of four subunits as follows: the
catalytic subunit; p125, which is tightly associated with the p50 subunit; ...p68, a proliferating cell nuclear antigen (PCNA)-binding
protein; and a fourth subunit, p12. In this study, the functional roles of the p12 subunit of pol δ were studied. The inter-subunit
interactions of the p12 subunit were determined by yeast two-hybrid assays and by pulldown assays. These assays revealed that
p12 interacts with p125 as well as p50. This dual interaction of p12 suggests that it may serve to stabilize the p125-p50
interaction. p12 was shown to be a novel PCNA-binding protein. This was confirmed by identification of a PCNA-binding motif
at its N terminus by binding assays and by site-directed mutagenesis. The activities and reaction products of recombinant
pol δ containing a p12 mutant defective in PCNA binding, as well as purified recombinant pol δ and its subassemblies, were
analyzed. Our results indicate that p12 contributes to PCNA-dependent pol δ activity, i.e. the p12-PCNA interaction is functional. Our data indicate that both p12 and p68 are required for optimal pol δ activity.
This supports the hypothesis that the interaction between pol δ and PCNA is a divalent one that involves p12 and p68. We propose
a model in which pol δ interacts with PCNA via at least two of its subunits, and one in which p12 could play a role in stabilizing
the overall pol δ-PCNA complex as well as pol δ itself.
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