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

---

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

Ultraviolet mutagenesis of the shuttle vector plasmid pZ189 in Xeroderma Pigmentosum cells yields a mutational pattern marked by hotspots at photoproduct sites on both strands of the supF marker gene. In order to test the influence of strand orientation on the appearance of hotspots the mutagenesis study was repeated on a vector with the supF gene in the inverted orientation. We recovered a pattern the same as that in the earlier work and conclude that the nature of the DNA polymerase involved in the replication of specific strands is not a primary determinant of hotspot occurrence in this system. One of the hotspots lies in an 8 base palindrome while the corresponding site on the other strand was not a hotspot. These results were obtained with calcium phosphate transfection of the UV treated vector. When DEAE dextran was used as a transfection agent both sites in the palindrome were hotspots. In a mixing experiment the calcium phosphate pattern was recovered. Our data suggest that the sequence determinants of mutational probability at these two sites lie outside the 8 bases of the palindrome and that mutagenesis at one, but not the other, site is sensitive to perturbation of cellular calcium levels.
...
PMID:Modulation of an ultraviolet mutational hotspot in a shuttle vector Xeroderma cells. 202 67

Aphidicolin inhibits purified DNA polymerases-a and -d in vitro and inhibits mitosis in animal cells. The Chinese hamster V79 cell mutant, Aphr-4-2, was selected for its ability to form colonies in cultured medium supplemented with 1.0 microM aphidicolin. At this concentration, the parental wild-type V79 cells (clone 743x) have a survival rate of less than 10(-7). The mutant DNA polymerase-a is resistant to aphidicolin at concentrations that are inhibitory to the wild-type V79 DNA polymerase-a. The apparent Km for dCTP of the mutant DNA polymerase-a is consistently lower than that of the wild-type DNA polymerase-a. This mutant exhibits slow growth, mutator activity, hypersensitivity, and hypermutability to UV. We wanted to know the basis of UV hypersensitivity in this mutant. Using the antisera (UV2) raised against UV-induced thymidine dimers and a sensitive immunofluorescence assay to measure UV-induced thymidine dimers and with detection in ACAS 570 Workstation, we observed that 50% of the thymidine dimers disappeared within 5 h after irradiation and more than 80% of the dimers were removed within 24 h in both cell lines. These results indicate that the recognition, incision, and excision steps in nucleotide excision repair pathway are normal in the mutant. In order to know if there is a difference in DNA polymerase-a or -d activities in the parental V79(wt) and Aphr-4-2 cells, DNA polymerases were partially purified from the parental and the mutant cells using sequential centrifugation and column chromatographies on DEAE-cellulose (DE23 and DE52) to remove DNA polymerases-beta and -gamma. More than 90% of the enzymatic activities from both cells showed characteristics of DNA polymerase-a type on the basis of these criteria: sensitivity to butyl phenyl dGTP (1 microM) and to IgG raised against DNA polymerase-a (SJK 132-20). The results indicate that DNA replication involving a mutant DNA polymerase-a with altered affinity for dCTP may be responsible for the UV sensitivity and mutability of the mutant.
...
PMID:On the DNA polymerase-a mutant: immunofluorescence assay of UV-induced thymidine dimers in Aphr-4-2 cells. 210 26

A stable DNA polymerase (EC 2.7.7.7) has been purified from the extremely thermophilic eubacterium Thermotoga sp. strain FjSS3-B.1 by a five-step purification procedure. First, the crude extract was treated with polyethylenimine to precipitate nucleic acids. The endonuclease activity coprecipitated. DEAE-Sepharose, CM-Sephrarose, and hydroxylapatite column chromatography were used to purify the preparation. As a final step on a small scale, preparative sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis was used. The purified DNA polymerase exhibited a molecular weight of 85,000, as determined by both SDS-polyacrylamide gel electrophoresis and size-exclusion chromatography. Its pH optimum was in the range pH 7.5-8. When assayed over the temperature range 30-80 degrees C, the maximum activity in a 30-min assay was at 80 degrees C. The enzyme was moderately thermostable and exhibited half-lives of 3 min at 95 degrees C and 60 min at 50 degrees C in the absence of substrate. Several additives such as Triton X-100 enhanced thermostability. During storage at 4 degrees C and -70 degrees C, the stability of the enzyme was improved by the addition of gelatin.
...
PMID:Purification and some properties of a thermostable DNA polymerase from a Thermotoga species. 227 6

The polynucleotide length of single-stranded regions in double-stranded DNA may be determined by caffeine gradient elution from benzoylated DEAE-cellulose. On the basis of this principle, analysis has been made of sheared, deproteinized DNA isolated from synchronized lymphoblastoid cells. Two classes of single-stranded regions were detected. A minor fraction of replicating DNA contained single-stranded regions of 200 nucleotides, whilst the major structural discontinuity involved single-stranded regions of 1-4 kilobases. Newly incorporated [3H]thymidine was principally associated with the latter. Using a 'pulse-chase' protocol, the effect of certain cytotoxic drugs (and related compounds) on the proportion of replicating DNA exhibiting single-stranded character was assessed. The effects were variable. The proportion was increased by hydroxyurea and 3-aminobenzamide, but decreased by inhibitors of DNA polymerase and, to a greater extent, by inhibitors of topoisomerase. Caffeine gradient elution associated drug-induced changes with the radiolabelling of long single-stranded regions. The results are consistent with models of DNA replication involving DNA polymerization remote from replicating forks.
...
PMID:Structural analysis of replicating DNA following exposure to cytotoxic drugs: implications for current models of DNA synthesis in mammalian cells. 231 56

The reverse transcriptase polymerase of the human T-cell lymphotropic virus/lymphadenopathy-associated virus has been cloned into an expression vector and expressed in Escherichia coli. Two polypeptides of 66 and 51 kDa molecular mass are detectable in polymerase-expressing bacterial lysates with human patient sera. They are processed from a short-lived 120-kDa polyprotein precursor equivalent to a region consisting of polymerase, protease, and endonuclease. The 51 kDa protein appears to originate from the 66-kDa molecule; additional processing products are 32- and 15-kDa proteins. The bacterially expressed polymerase is enzymatically active and exhibits the template specificities, ion requirements, and response to inhibitors of the authentic enzyme. It was purified by DEAE-cellulose-, phosphocellulose-, and poly(rC)-agarose column chromatography followed by glycerol density gradient centrifugation. It copurifies with an RNase H activity, suggesting the existence of a virus-coded DNA polymerase-RNase H complex. The purified bacterial enzyme allows a safe large-scale screening for inhibitors of both activities.
...
PMID:RNase H activity associated with bacterially expressed reverse transcriptase of human T-cell lymphotropic virus III/lymphadenopathy-associated virus. 244 62

Polyclonal antibodies responding specifically to the N-terminal, central and C-terminal polypeptide domains of the herpes simplex virus type I (HSV-1) DNA polymerase of strain Angelotti were generated. Each of the five different rabbit antisera reacted specifically with a viral 132 +/- 5-kDa polypeptide as shown by immunoblot analysis. Enzyme binding and inhibition studies revealed that antibodies raised to the central and the C-terminal domains of the protein inhibited the polymerizing activity by 70-90%, respectively, which is well in line with the proposed site of the catalytic center of the enzyme and with the possible involvement of these polypeptide chains in DNA-protein interactions. In agreement with this, antibodies directed towards the N-terminal domain bound to the enzyme without effecting the enzymatic activity. The strong binding but low inhibitory properties of antibodies directed to the polypeptide region between residues 1072 and 1146 confirms previous suggestions that these C-terminal sequences, which share no homology to the Epstein-Barr virus DNA polymerase, are less likely involved in the building of the polymerase catalytic site. Antibodies, raised to the very C terminus of the polymerase (EX3), were successfully used to identify a single 132 +/- 5-kDa polypeptide, which coeluted with the HSV DNA polymerase activity during DEAE-cellulose chromatography, and were further shown to precipitate a major viral polypeptide of identical size. From the presented data it can be concluded that the native enzyme consists of a single polypeptide with a size predicted from the long open reading frame of the HSV-1 DNA polymerase gene.
...
PMID:The herpes simplex virus type I DNA polymerase. Polypeptide structure and antigenic domains. 245 39

Two species of DNA polymerase alpha free of primase activity were identified in extracts of Ehrlich mouse cells that had been infected with minute virus of mice. Primase-free forms of DNA polymerase alpha eluted with 150 and 180 mM NaCl during ion-exchange chromatography on DEAE-cellulose columns, exhibited sedimentation coefficients of 11 S and 8.2 S, respectively, and were inhibited by aphidicolin, N2-(p-n-butylphenyl)-9-(2-deoxy-beta-D-ribofuranosyl)guanine 5'-triphosphate, and 2-(p-n-butylanilino)-9-(2-deoxy-beta-D-ribofuranosyl)adenine 5'-triphosphate. The ratio of primase-free DNA polymerase alpha to the DNA polymerase alpha-primase complex increased from 1.5 to greater than 100 during the course of infection, and free primase was produced during the MVM replicative cycle.
...
PMID:A novel primase-free form of murine DNA polymerase alpha induced by infection with minute virus of mice. 254 83

Japanese quail oviduct total cDNA was synthesized from total mRNA by the classical method, using AMV reverse transcriptase, the Klenow fragment of DNA polymerase I and S1 nuclease. The results of the synthesis of total ss-cDNA partially differed from those published for the synthesis of chicken total ss-cDNA. The presumed causes of the differences in the complete reverse transcription of mRNAcon and incomplete reverse transcription of mRNAlys and a large part of mRNAov in our case are discussed. An atypical strategy was used for cloning full-length cDNAov. The total cDNA was dC-tailed, then fractionated by analytical agarose electrophoresis and 4 cDNA fractions of different lengths were isolated from the gel using DEAE cellulose membranes. A cDNA fraction about 1500-2500 bp long containing full-length cDNAov was annealed with dG-tailed PstI-linearized plasmid pBR322 and cloned into competent E. coli DHl cells. Seventy-two clones were screened for the presence of full-length cDNAov, initially by insert size and then by means of hybrid-arrested translation. Four clones containing 1900-1980 bp cDNAov were obtained. The cDNA ends in one of these clones were sequenced. Comparison of these sequences with those of chicken mRNAov indicated that almost full-length cDNAov's had been cloned. They lacked a small number of nucleotides at their 5' ends, which had probably been split off during the degradation of the hairpin loop by S1 nuclease. A sequence of 134 bases from the 5' end of mRNAov is presented and compared with the known sequence of chicken mRNAov. The advantages of the cloning strategy employed, in particular, its cloning efficiency and the possibility of simultaneously identifying clones of also other oviduct cDNA species (in this work: cDNAY and, tentatively, cDNAcon), are discussed.
...
PMID:Cloning of Japanese quail ovalbumin cDNA in E. coli. 265 90

A preparation of human placenta DNA polymerase with specific activity 6000 unit/mg was obtained. The protocol of the enzyme purification includes the crude extract preparation, the subsequent chromatographies on phosphocellulose, red sepharose, DEAE sepharose and hydroxylapatite. The isolated DNA polymerase belongs to alpha-type according to the large molecular mass (greater than 150 kDa), high sensitivity to N-ethylmaleimide, the profound inhibition of DNA polymerization activity by 200 mM KCl and the ability to catalyze DNA synthesis, using the deoxyribonucleic template and ribonucleic primer. The DNA polymerase preparations contain a few forms with Stokes radii 50-60 A and sedimentation coefficients 7.3-9.0 S as found from data of gel-filtration and ultracentrifugation in glycerol density gradient, accordingly. The existence of four various forms of DNA polymerase activity: 150, 170, 220, 480 kDa were revealed by native electrophoresis. The four steps of purification result in DNA polymerase preparation that was shown by electrophoresis to contain 15-20% of protein possessing the polymerase activity. However the preparation obtained seems to be a "chromatographically pure substance", according to following ion-exchange and affinity chromatographies. The other proteins without polymerase activity are suggested to be the components of the replicative complex of human placenta cells.
...
PMID:[Purification and characterization of DNA-dependent DNA-polymerase alpha from human placenta]. 277 Jul 23

Cis-platinum derivatives were observed to inhibit the activity of DNA polymerase alpha of P388 lymphocytic leukemia cells. A 600g nuclear preparation of the polymerase alpha was inhibited by cis-diamminedichloroplatinum(II) [cDDP], diamminemalonatoplatinum(II) [MAL], (1,2-diaminocyclohexane)-dichloroplatinum(II) [DACH-Pt-CL2], and (1,2-diaminocyclohexane)malonato-platinum(II) [DACH-Pt-MAL]. cDDP was a more potent inhibitor of the enzyme activity which was positively correlated with the observed inhibition of DNA synthesis of P388 cells in vivo and in vitro. The inhibition of the 600g preparation by cDDP could be partially reversed by the addition of exogenous ctDNA, but 35% inhibition was not retreivable by adding new template. Isolation of the P388 DNA polymerase alpha enzyme by DEAE column chromatography led to an enzyme with 100 fold purification, which was sensitive to N-ethyl maleimide at 0.1 mM concentration. cDDP inhibited the activity of this enzyme in a dose dependent manner. However, MAL, DACH-Pt-Cl2 and DACH-Pt-MAL afforded no inhibition, nor did the latter two derivatives bind to the enzyme. cDDP inhibition of the activity of purified enzyme was partially reversed by the addition of exogenous ctDNA and by the addition of dGTP, whereas addition of other d(NTP)s had no effect on the recovery of the enzyme activity. These studies suggest that cDDP inhibits DNA polymerase alpha activity and that the inhibition is not the sole mechanism of the action of the drug in suppression of DNA synthesis and cell death. Preliminary studies suggest that the drug may bind to the apoprotein of the enzyme in a competitive manner with dGTP.
...
PMID:The inhibition of P338 lymphocytic leukemia DNA polymerase alpha activity by cis-diamminedichloroplatinum(II) and related derivatives. 281 17


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>

---