Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.7 (DNA polymerase)
17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The papovavirus SV40 is able to induce tumours in susceptible hosts and will transform cells in vitro. Its major early protein, large T antigen, is required for viral DNA synthesis, both in vivo and in vitro, and is also responsible for the oncogenic action of the virus. We have made use of an extensive library of anti-T monoclonal antibodies to investigate the cellular effects of T. Large T shares an antigenic determinant with a growth-regulated host protein, p68, which is a member of an expanding super-family of helicases with particular homology to the translation initiation factor elF-4A. We have also studied the binding and interaction of large T with two particular host components: the replicative enzyme DNA polymerase alpha and the proto-oncogene p53. These two proteins bind to similar regions of T and exert similar effects on its antigenic structure. We found that p53 can block the binding of DNA polymerase alpha to T as well as co-existing with DNA polymerase alpha in a trimeric complex with T. This suggests that these interactions may be important in the oncogenic and replicative action of large T.
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PMID:Host proteins that bind to or mimic SV40 large T antigen: using antibodies to look at protein interactions and their significance. 247 59

An efficient in vitro system for generating soluble complexes between simian virus 40 T antigen and the cellular protein p53 was developed. A p53 cDNA was inserted 3' to the SP6 promoter in pGEM-1 (Promega-Biotec) and transcribed by SP6 polymerase. In vitro translation of the cRNA generated p53 which was immunoprecipitable with all five monoclonal antibodies tested (PAb122, PAb421, PAb242, PAb246, and PAb248). The p53 sedimented at about 8-10 S in sucrose gradients, possibly corresponding to a tetramer. T-antigen-p53 complexes were produced by the addition of immunoaffinity-purified T antigen to p53-containing translation lysates. Equivalent amounts of p53 were immunoprecipitated with the anti-T-antigen antibodies PAb416, PAb419, and PAb101, suggesting that in vitro made p53 complexed mostly to a population of T-antigen molecules that had matured at least 15 min in the cell. The complexes sedimented at 18-20 S in sucrose gradients. In order to map the p53 binding site on T antigen, p53 was complexed in vitro to labeled proteolytic fragments of T antigen. A 46K fragment, spanning residues 131-517, was immunoprecipitated with the anti-p53 monoclonal PAb122 and therefore is likely to contain the p53 binding site. This region contains T-antigen sequences necessary for the efficient transformation of nonpermissive cells and for the induction of cellular rRNA synthesis. It also contains the binding sites for DNA polymerase alpha and ATP. We suggest a possible role for T-p53 complexes in T-antigen-associated functions.
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PMID:Characterization of the in vitro interaction between SV40 T antigen and p53: mapping the p53 binding site. 283 65

The large T antigen (T) of simian virus 40 is a multifunctional protein required for both viral DNA replication and cellular transformation. T antigen forms specific protein complexes with the host protein p53 in both virus-infected and transformed cells. p53 has recently been shown to be an oncogene, but its normal function is not clear. We previously established a radioimmunoassay to study the newly described complex between T antigen and DNA polymerase alpha, and have noted a similarity between the antigenic changes induced in T by the binding of both p53 and polymerase. We now extend this analysis to a larger collection of anti-T antibodies and formally establish that p53 and DNA polymerase alpha can compete for binding to the SV40 T antigen. At a critical concentration of the three components it is possible to detect a trimeric complex of T, p53 and DNA polymerase alpha. Our observations have important implications for the control by these nuclear oncogenes of viral and cellular DNA synthesis and viral host range in both normal and transformed cells. We present a model for the action of p53 in growth control.
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PMID:p53 and DNA polymerase alpha compete for binding to SV40 T antigen. 330 72

Adenoviral vectors have recently been shown to effectively deliver genes into a variety of tissues. Since these vectors have some advantages over the more extensively investigated retroviruses, we studied the effect of two replication-defective adenovectors bearing human wild type tumor suppressor gene p53 (Adp53) and Escherichia coli beta-galactosidase gene (AdLacZ) on 9L glioma cells. Successful in vitro gene transfer was shown by DNA polymerase chain reaction (PCR), and expression was confirmed by reverse transcriptase RNA PCR and Western blot analyses. Transduction of 9L cells with the Adp53 inhibited cell growth and induced phenotypic changes consistent with cell death at low titers, while AdLacZ caused cytopathic changes only at high titers. Stereotactic injection of AdLacZ (10(7) plaque forming units) into tumor bed stained 25 to 30% of tumor cells at the site of vector delivery. Injection of Adp53 (10(7) plaque forming units), but not AdLacZ (controls), into established 4-day old 9L glioma brain tumors decreased tumor volume by 40% after 14 days. As a step toward gene therapy of brain tumors using replication-defective adenoviruses, these data support the use of tumor suppressor gene transfer for in vivo treatment of whole animal brain tumor models.
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PMID:Adenovirus-mediated p53 gene delivery inhibits 9L glioma growth in rats. 764 77

DNA polymerase fingerprint analysis (DPFA) was employed for identifying DNA-carcinogen adduct formation in the human p53 and lac gene sequence. Two 'hot regions' at codons 223-250 and 257-283 of the p53 gene were easily attacked by nitroso-2-acetylaminofluorene or acetoxy-2-acetylaminofluorene. However, the promutational lesions in lac gene were rather randomly distributed. The chemical treated plasmid (pUC 19) which contains lac gene were transfected into Escherichia coli JM109 cells and the induced lac gene mutants were selected with X-Gal plate as indicated by the appearance of white colonies. No mutational hot regions were found in the lac gene.
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PMID:Preferential promutagenic lesions at exons 7-8 of human p53 genomic DNA induced by the direct-acting hepatocarcinogens N-nitroso-2-acetylaminofluorene and N-acetoxy-2-acetylaminofluorene. 765 42

Denaturing gradient gel electrophoresis (DGGE) separates (DNA) molecules based on their sequence. Using the proper conditions, all base-pair substitutions can be resolved from the wild-type sequence using DGGE. Polymerase chain reaction (PCR) permits rapid amplification of a given region of the genome. In this paper, we demonstrate the utility of DGGE combined with PCR for mutation analysis by presenting different examples: (i) analysis of mouse p53 cDNA for mutations, (ii) simultaneous analysis of thousands of 4NQO-induced mutants for mutations in HPRT exon 3, (iii) examination of the fidelity of the thermostable DNA polymerase isolated from Pyrococcus furiosus (Pfu), (iv) purification of mutant DNA from contaminating wild-type DNA from mouse spleenic T-cell clones.
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PMID:Mutational analysis using denaturing gradient gel electrophoresis and PCR. 768 54

The propensity of a cell to undergo apoptosis has been proposed to be a determinant for chemotherapy sensitivity that is not directly dependent on specific drug-target interactions. Androgen-independent prostate cancer is typically refractory to cytotoxic drugs, and we tested whether this is due to a loss of the ability to undergo apoptosis. Exposure of the hormone-insensitive and p53-negative human prostate carcinoma cell line PC-3 to 22 microM cisplatin, 1 microM camptothecin, 10 microM tenoposide, 135 nM vincristine, or 10 microM lovastatin for 72 h caused cell death, internucleosomal DNA fragmentation, and morphological changes typical for apoptosis. One microM cycloheximide prevented anticancer drug-induced apoptosis, whereas high concentration (1 mM) of cycloheximide alone induced apoptosis, indicating that protein synthesis was not needed for these cells to undergo apoptosis. Since cycloheximide affected DNA synthesis and proliferation of PC-3 cells, we tested whether the DNA polymerase inhibitor aphidicolin could also suppress drug-induced apoptosis. In contrast to cycloheximide, aphidicolin inhibited only vincristine-induced apoptosis. Cycloheximide prevented drug-induced changes in cell cycle distribution except for vincristine, while aphidicolin led to an accumulation of cells at the G1-S border independent of the drug used. These data indicate that macromolecular synthesis, active cell cycling, and p53 expression are not required for apoptosis to proceed in this system.
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PMID:Drug-induced apoptosis is not necessarily dependent on macromolecular synthesis or proliferation in the p53-negative human prostate cancer cell line PC-3. 774 12

With DNA polymerase chain reaction-single strand conformation polymorphism assay followed by direct DNA sequencing, p53 gene mutation was examined in bladder transitional epithelial cell carcinoma, renal cell carcinoma and testicular seminoma. p53 gene mutation was found in 7 cases (35%) of bladder carcinoma and 4 cases (23.5%) of testicular seminoma. Inactivation of Rb gene and activation of ras and c-erbB-2 were also studied. The results suggest that development of urologic neoplasms is closely associated with p53 gene mutation and involves loss of expression of Rb and aberrant expression of ras and c-erbB-2.
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PMID:[Mechanism of p53 gene mutation in the development of urologic cancer]. 786 97

A plasmid carrying the 5' flanking region of the mouse proliferating-cell-nuclear-antigen (PCNA) gene or DNA polymerase beta gene was fused with the chloramphenicol acetyltransferase (CAT) gene, then cotransfected into mouse N18TG2 cells with the expression plasmid for the p53 gene. Expression of the wild-type p53 repressed the CAT expression directed by the PCNA gene promoter, while it had little effect on the DNA polymerase beta gene promoter. RNase protection analysis revealed that the repression of the PCNA gene promoter by p53 was at the transcription step. Analysis with various deletion mutants in the PCNA gene promoter revealed that a specific sequence is not required for the repression, suggesting that p53 represses the PCNA gene promoter by interacting with some components of the basic transcription machinery. By analysis with various deletion mutants in the DNA polymerase beta gene promoter, we identified the unique 10-bp palindromic sequence (-24 to -15), in the presence of which p53 was not able to repress the promoter activity. This sequence conferred resistance to p53 repression onto the PCNA gene promoter, when it was placed 21-bp upstream from the transcription-initiation site.
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PMID:Differential effect of p53 on the promoters of mouse DNA polymerase beta gene and proliferating-cell-nuclear-antigen gene. 790 18

The p53 tumour-suppressor protein controls the expression of a gene encoding the p21 cyclin-dependent protein kinase (CDK) regulator. Levels of p21 protein are increased in senescent cells and p21 overexpression blocks the growth of tumour cells. In normal human cells, but not in many tumour cells, p21 exists in a quaternary complex with a cyclin, a CDK, and the proliferating-cell nuclear antigen (PCNA). p21 controls CDK activity, thereby affecting cell-cycle control, whereas PCNA functions in both DNA replication and repair. Here we use simian virus 40 DNA replication in vitro to show than p21 directly inhibits PCNA-dependent DNA replication in the absence of a cyclin/CDK. Furthermore, p21 blocks the ability of PCNA to activate DNA polymerase delta, the principal replicative DNA polymerase. This regulation results from a direct interaction between p21 and PCNA. Thus, during p53-mediated suppression of cell proliferation, p21 and PCNA may be important for coordinating cell-cycle progression, DNA replication and repair of damaged DNA.
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PMID:The p21 inhibitor of cyclin-dependent kinases controls DNA replication by interaction with PCNA. 791 Dec 27


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