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)

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

Previously we cloned and sequenced the polC gene of Bacillus subtilis and identified regions corresponding to various catalytic domains of DNA polymerase III, the enzyme it encodes. In the present study, by using primer extension, we have identified the transcription start site and a 139 nucleotide leader region upstream of the first codon. This region contains a DnaA box in the non-transcribed DNA strand. An RNA transcript of the leader would contain a sequence that could form a 29 bp stem-loop secondary structure followed by a strong terminator sequence, rich in uracil, before the ribosome binding site. Plasmids were constructed containing either the intact leader region or deletion mutations of the leader, fused to the Escherichia coli lacZ gene in an expression vector. Single copies of the fusions were then integrated into the B. subtilis genome by transformation. Studies of the expression of beta-galactosidase by the transformed cells supported the idea that the leader region is important in regulating polC gene expression.
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PMID:Leader region of the gene encoding DNA polymerase III of Bacillus subtilis. 767 75

The genomic localization of two immunodominant genes encoding two proteins of the Epstein-Barr virus capsid antigen (VCA) complex, VCA-p18 and VCA-p40, has been identified. For that purpose, lambda gt11-based cDNA libraries were constructed from HH514.c16 cells induced for virus production. The libraries were screened with a monoclonal antibody, EBV.OT41A, directed against VCA-p40 or with affinity-purified human antibodies against VCA-p18. Sequencing of the inserts of positive plaques showed that VCA-p18 and VCA-p40 are encoded within open reading frames (ORFs) BFRF3 and BdRF1, respectively. Peptide scanning analysis of the predicted protein of ORF BdRF1 resulted in defining the epitope of monoclonal antibody EBV.OT41A at the C-terminal region. The dominant VCA-p18 reactivity of human sera can be completely inhibited by preadsorption with Escherichia coli-expressed BFRF3-beta-galactosidase. Serum of a rabbit immunized with BFRF3-beta galactosidase reacts with a VCA-specific protein of 18 kDa. In addition, BFRF3-beta-galactosidase affinity-purified antibodies react with VCA-p18 of virus-producing cells (HH514.c16). Complete inhibition of viral DNA polymerase activity by phosphonoacetic acid is associated with the absence of RNAs and protein products of both ORFs, indicating that VCA-p18 and VCA-p40 are true late antigens.
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PMID:Gene mapping and expression of two immunodominant Epstein-Barr virus capsid proteins. 768 3

Molecular genetic studies have revealed that the human hepatitis B viral (HBV) Pol protein, a polypeptide of about 94 kDa, contains four domains. These are the 5'-terminal protein, spacer, RNA reverse transcriptase/DNA polymerase, and RNase H, respectively, from the amino (N) to carboxy (C) terminus. No evidence indicates as yet the involvement of a specific protease in cleaving the Pol protein or the presence of protease-cutting sites in the Pol protein. An in vitro-translated Pol protein was shown to be cleaved by purified thrombin but not in the presence of its inhibitor, hirudin. Two thrombin-cutting sites, spanning 194 amino acids, were then deduced by thrombin digestion of Pol protein with various lengths of C-terminal deletion. These two putative cutting sites, one located in the spacer region and the other in the beginning of the polymerase region, were found to be conserved at similar positions in the Pol protein of all hepadnaviruses. By using a novel method called the LacZ localization assay (LLA), it was demonstrated that a tripartite fusion protein containing the nucleus localization sequence (NLS) of SV40 large T Ag, the putative thrombin cutting sequence (Ile-Arg-Ile-Pro-Arg320-Thr) of HBV Pol protein and the full length beta-galactosidase of E. coli, exhibited a lower percentage (approximately 53%) of targeting into the nucleus of transfected hepatoma cells when compared with a similar tripartite protein containing a single mutation (Arg320 residue into Trp320) of HBV Pol protein (approximately 78%) or with a bipartite protein of SV40 NLS-beta-galactosidase (approximately 90%). These results indicate that the putative thrombin-cutting site in the spacer region of HBV Pol protein could be cleaved by a cellular protease resulting in the separation of NLS sequence from the beta-galactosidase and rendering a lower frequency of X-gal staining in the nucleus.
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PMID:Demonstration of the presence of protease-cutting site in the spacer of hepatitis B viral Pol protein. 773 Apr 38

We isolated a murine gene for the DNA polymerase alpha associated protein P1, which shares high homology with the budding yeast MCM3 protein, which is a member of a protein family involved in the early event of DNA replication having a putative DNA-dependent ATPase motif. Using a polyclonal anti-P1 antibody raised against a beta-galactosidase-P1 fusion protein, we identified at least two forms of P1 protein in the nucleus of a mouse cell line, an underphosphorylated form that was associated with a particular nuclear structure and a hyperphosphorylated form loosely bound to the nucleus. During progression through S phase, P1 disappeared, first from the euchromatic region, then from the heterochromatic region, apparently in parallel with temporally ordered DNA replication. Thus, it is likely that the underphosphorylated P1 is dissociated from the nuclear structure after DNA replication by cell cycle-dependent phosphorylation. This is the first direct observation of a protein whose behavior is consistent with that of a hypothetical factor which restricts the chromatin to replicate once per cell cycle in higher eukaryotes.
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PMID:DNA polymerase alpha associated protein P1, a murine homolog of yeast MCM3, changes its intranuclear distribution during the DNA synthetic period. 792 75

In order to study the conversion of UV lesions into frameshift and base substitution mutations, M13mp2 phage DNA was altered by the addition of extra pyrimidines, or by construction of a nonsense codon preceded by a run of pyrimidines within the beta-galactosidase complementing region. The normal sequence 5' GTC GTT TTA CAA 3' was changed to GTC GTT T TTA CAA (MIDT) or GTC GTT C TTA CAA (MIDC) to study frameshifts and to GTC GTT CTT TAA (OCHRE) to study reversion of the ochre (TAA) codon. Escherichia coli pol I Kf and T7 DNA polymerase mutant enzymes devoid of 3'-->5' exonuclease activity produced UV-induced revertants at higher frequency than did their exonuclease proficient counterparts. Removal of cyclobutane dimers with photolyase before in vitro synthesis did not greatly affect mutant frequency although such treatment led to significantly increased DNA synthesis by the wild-type T7 DNA polymerase on UV-irradiated substrate. Reversions of the in frame ochre sequence GTT CTT TAA produced by the delta 28 T7 DNA polymerase were mainly by base substitution in the TAA codon. About half of the E. coli Kf exo- enzyme ochre revertants had a TTA deletion. Five mutant T7 DNA polymerases with varying exonuclease activity gave revertant frequencies that correlated better with published values of enzyme velocity than with exonuclease activity or with measured bypass synthesis. Our data indicate that loss of proofreading activity increases the frequency of UV-induced frameshifts, but lack of such activity is not sufficient for their production. We suggest that frameshifts occur more frequently when nucleotide addition opposite the lesion is slow. The same lesion can give rise to a different spectrum of mutations depending on the polymerase.
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PMID:Production of UV-induced frameshift mutations in vitro by DNA polymerases deficient in 3'-->5' exonuclease activity. 802 6

Oxygen free radicals are produced in large amounts by normal cellular processes. Damage to DNA by these reactive species has been implicated in mutagenesis and may be important in the etiology of a variety of human diseases. In this study we investigate the types of mutations produced in vitro as a result of DNA damage by oxygen free radicals. We used a lacZ alpha forward mutation assay in which M13 viral DNA is damaged in vitro, replicated with purified DNA polymerase alpha or beta, transfected into E. coli, and screened for mutations by reduced alpha-complementation of beta-galactosidase activity. By determining the effects of damaged templates on the fidelity of individual DNA polymerases involved in replication and repair, we address the role of specific DNA polymerases in mutagenesis induced by reactive oxygen species. Aerobic incubation of DNA with 100 microM CuCl, 10 microM H2O2 and 100 microM ascorbic acid results in a 3.3-fold and a 3.6-fold elevation in mutation frequency for polymerases alpha and beta, respectively. The specificity and location of the induced mutations, however, are entirely different. For polymerase alpha, A to C, and C to A transversions and deletions of C are each elevated more than 10-fold over their frequencies on undamaged template. For polymerase beta, A to T, C to T, C to A, G to C, and G to T substitutions, and deletions of G are elevated by damage. The frequency of mutants containing two or more closely spaced substitutions is also markedly increased by template damage although the types of mutations and their positions are again specific to each DNA polymerase. We conclude that, for oxidative lesions, the frequency and the types of mutations are determined in part by the DNA polymerase that encounters the site of damage.
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PMID:Oxygen radical induced mutagenesis is DNA polymerase specific. 828 53

Reactive oxygen species, produced in cells by a variety of mechanisms, damage DNA and cause mutations. To characterize the types of mutations produced in mammalian cells, we copied DNA damaged by reactive oxygen species with mammalian DNA polymerase beta. Double-stranded circular M13mp2 DNA containing a 361-nucleotide single-stranded gap within the lacZ gene was damaged by aerobic incubation with Fe2+ and H2O2. The gap then was filled by purified recombinant rat DNA polymerase beta, and the DNA was transfected into Escherichia coli. Mutations within the nonessential lacZ gene for beta-galactosidase were identified by reduced alpha-complementation. In this system, oxidative damage increased the mutation frequency within the target region by an average of 4.3-fold. At certain sites, the base substitution rate is nearly 300 times greater than would be expected to result from a random distribution of damage. The oxidatively induced mutations fall into two categories: those apparently caused by direct miscoding of modified DNA and those associated with enhanced misincorporation at prexisting polymerase-specific hot spots. The latter group may be due to a conformational change in the DNA caused by oxidative modification and could be indicative of a novel mutagenic mechanism.
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PMID:Mechanisms of mutation by oxidative DNA damage: reduced fidelity of mammalian DNA polymerase beta. 847 71

The region between the rpmH and dnaA genes contains five promoters that divergently express the ribosomal protein L34 and the proteins of the dnaA operon, including DnaA, the beta clamp of DNA polymerase III holoenzyme, and RecF. The DNA-binding protein Fis was shown by the band shift assay to bind near the rpmHp2 and dnaAp2 promoters and by DNase I footprinting to bind to a single site in the dnaAp2 promoter overlapping the -35 and spacer sequences. There were no observable differences in Fis affinity or the angle of bending induced by Fis between methylated and unmethylated DNA fragments containing the Fis binding site in the dnaAp2 promoter. Fis directly or indirectly represses the expression of DnaA protein and the beta clamp of DNA polymerase III. A fis null mutant containing a dnaA-lacZ in-frame fusion had twofold greater beta-galactosidase activity than a fis wild-type strain, and induced expression of Fis eliminated the increase in activity of the fusion protein. A two- to threefold increase in the levels of DnaA and beta clamp proteins was found in a fis null mutant by immunoblot gel analysis.
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PMID:Fis binding in the dnaA operon promoter region. 883 Jun 99

The expression of genes involved in DNA replication is closely correlated with the proliferating state of cells and is repressed with the progression of differentiation during development. Promoter regions of the Drosophila proliferating cell nuclear antigen (PCNA) gene and the DNA polymerase alpha gene contain a common 8-base pair promoter element (DRE: DNA replication-related element). The examination of a common expression mechanism for DNA replication-related genes, which is regulated positively by growth signals and negatively by differentiation signals would be of interest. We generated PCNA-LacZ fusion genes in which the 5'-flanking sequence of the PCNA gene has been mutated. An examination of the expression of these fusion genes, introduced into flies by germ-line transformation, led to the identification of another distinct regulatory element, URE (upstream regulatory element), within the region from -168 to -119 with respect to the transcription initiation site. During embryogenesis, the region containing the DRE sequence (-108 to -91) greatly stimulated the PCNA gene minimal promoter (-86 to +130), when it was placed upstream of the promoter in both normal and reverse orientations. Addition of the URE sequence further stimulated the promoter activity twofold. During larval stages, both DRE and URE were indispensable to the promoter activity, since neither of the sequences alone activated the minimal promoter. Demonstration of beta-galactosidase activity indicated URE plays an essential role in various larval tissues such as salivary gland and imaginal disc. While the minimal promoter region alone directed maternal expression of lacZ in ovaries of adult females, both DRE and URE further stimulated promoter activity. These results show several elements of the PCNA gene promoter play roles during Drosophila development.
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PMID:Roles of multiple promoter elements of the proliferating cell nuclear antigen gene during Drosophila development. 907 66


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