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 structural gene for DNA polymerase II was cloned by using a synthetic inosine-containing oligonucleotide probe corresponding to 11 amino acids, which were determined by sequencing the amino terminus of the purified protein. The labeled oligonucleotide hybridized specifically to the lambda clone 7H9 from the Kohara collection as well as to plasmid pGW511 containing the SOS-regulated dinA gene. Approximately 1400 base pairs of dinA sequence were determined. The predicted amino-terminal sequence of dinA demonstrated that this gene encoded DNA polymerase II. Sequence analysis of the upstream region localized a LexA binding site overlapping the -35 region of the dinA promoter, and this promoter element was found to be only two nucleotides downstream from the 3' end of the araD gene. These results demonstrate that the gene order is thr-dinA (pol II)-ara-leu on the Escherichia coli chromosome and that the DNA polymerase II structural gene is transcribed in the same direction as the araBAD operon. Based on the analysis of the predicted protein, we have identified a sequence motif Asp-Xaa-Xaa-Ser-Leu-Tyr-Pro-Ser in DNA polymerase II that is highly conserved among a diverse group of DNA polymerases, which include those from humans, yeast, Herpes and vaccinia viruses, and phages T4 and PRD1. The demonstration that DNA polymerase II is a component of the SOS response in E. coli suggests that it plays an important role in DNA repair and/or mutagenesis.
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PMID:DNA polymerase II is encoded by the DNA damage-inducible dinA gene of Escherichia coli. 221 98

Changes in the expression pattern of DNA polymerase beta gene during rat lung, brain, and testis development have been investigated. A decrease in the level of beta-pol mRNA was observed during postnatal development of lung and brain. By contrast, an almost 20-fold increase in the level of beta-pol mRNA was observed during spermatogenesis. For most adult rat tissues the abundance of beta-pol mRNA was low compared with that of beta-actin mRNA. Northern blot analysis revealed four distinct transcripts hybridizing to beta-pol probes. At least two of them, 1.4 kb and 4.0 kb, were products of a beta-polymerase gene. The changes in the expression pattern during lung and brain development, and during spermatogenesis, suggest involvement of DNA polymerase beta in gap-filling DNA synthesis during recombination.
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PMID:Changes in the DNA polymerase beta gene expression during development of lung, brain, and testis suggest an involvement of the enzyme in DNA recombination. 222 50

6-(p-Hydroxyphenylhydrazino)uracil (H2-HPUra) is a selective and potent inhibitor of the replication-specific class III DNA polymerase (pol III) of Gr+ bacteria. Although formally a pyrimidine, H2-HPUra derives its inhibitory activity from its specific capacity to mimic the purine nucleotide, dGTP. We describe the successful conversion of the H2-HPUra inhibitor prototype to a bona fide purine, using N2-(benzyl)guanine (BG) as the basis. Structure-activity relationships of BGs carrying a variety of substituents on the aryl ring identified N2-(3,4-dichlorobenzyl)guanine (DCBG) as a nucleus equivalent to H2-HPUra with respect to potency and inhibitor mechanism. DCBdGTP, the 2'-deoxyribonucleoside 5'-triphosphate form of DCBG, was synthesized and characterized with respect to its action on wild-type and mutant forms of B. subtilis DNA pol III. DCBdGTP acted on pol III by the characteristic inhibitor mechanism and formally occupied the dNTP binding site with a fit which permitted its polymerization.
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PMID:Development of novel inhibitor probes of DNA polymerase III based on dGTP analogs of the HPUra type: base, nucleoside and nucleotide derivatives of N2-(3,4-dichlorobenzyl)guanine. 225 29

We have studied antibodies (anti-pol antibody) against the polymerase gene product of hepatitis B virus by solid-phase enzyme immunoassay using synthetic peptides coded for by this gene. Sera from six patients with acute hepatitis B, 112 chronic hepatitis B virus carriers and six healthy individuals with naturally acquired immunity to hepatitis B virus were tested for anti-pol antibody. In acute hepatitis B virus infection, anti-pol antibody was detected in three of six patients. In chronic hepatitis B virus infection, anti-pol antibody was detected in 17 of 29 (59%), in 23 of 33 (70%) of cirrhotic patients and in 18 of 24 (75%) patients with cirrhosis complicated by hepatocellular carcinoma, compared with 4 of 19 (21%) asymptomatic carriers and 2 of 7 (29%) patients with chronic persistent hepatitis. Titers of anti-pol antibody were higher in cirrhotic patients with and without hepatocellular carcinoma than in patients with chronic active hepatitis. The presence of anti-pol antibody, however, had no relationship with hepatitis B virus-associated DNA polymerase activities and other viral replicative markers. As for sera from six healthy individuals with naturally acquired immunity to hepatitis B virus, two (33%) were positive for anti-pol antibody. These results indicate that the immune response toward the polymerase gene product is induced during acute and chronic hepatitis B virus infection. In chronic hepatitis B virus infection, anti-pol antibody may serve as a new marker indicative of a long period of hepatitis B virus-induced hepatitis.
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PMID:Detection of antibodies against the polymerase gene product in hepatitis B virus infection. 239 Oct 62

Synthesis of (p)ppRNA-DNA chains by purified HeLa cell DNA primase-DNA polymerase alpha (pol alpha-primase) was compared with those synthesized by a multiprotein form of DNA polymerase alpha (pol alpha 2) using unique single-stranded DNA templates containing the origin of replication for simian virus 40 (SV40) DNA. The nucleotide locations of 33 initiation sites were identified by mapping G*pppN-RNA-DNA chains and identifying their 5'-terminal ribonucleotide. Pol alpha 2 strongly preferred initiation sites that began with ATP rather than GTP, thus frequently preferring different initiation sites than pol alpha-primase, depending on the template examined. The initiation sites selected in vitro, however, did not correspond to the sites used during SV40 DNA replication in vivo. Pol alpha 2 had the greatest effect on RNA primer size, typically synthesizing primers 1-5 nucleotides long, while pol alpha-primase synthesized primers 6-8 nucleotides long. These differences were observed even at individual initiation sites. Thus, the multiprotein form of DNA primase-DNA polymerase alpha affects selection of initiation sites, the frequency at which the sites are chosen, and length of RNA primers.
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PMID:Selection of template initiation sites and the lengths of RNA primers synthesized by DNA primase are strongly affected by its organization in a multiprotein DNA polymerase alpha complex. 242 60

To detect the proliferating cells in situ, a monoclonal antibody against human DNA polymerase alpha (pol alpha) was employed because this enzyme is known to be present in the nucleus of the cells in G1, S, and G2 phases. In addition, the surface phenotype of pol alpha-positive proliferating lymphocytes in diseased lymph nodes was determined by double staining consisting of immunoperoxidase and immunoalkaline phosphatase methods with various monoclonal antibodies against lymphocyte membrane antigens. In the paracortical area of lymph nodes with reactive changes, proliferating cells were 17% or less, and most of them were helper T-cells, although suppressor T-cells and B-cells also proliferate to a certain extent. In contrast, the proliferating cell population in malignant lymphomas was generally more than 40%, and it showed a single surface phenotype, indicating monoclonal proliferation. In addition, an unusual T-cell antigen phenotype of proliferating cells was observed in some cases of peripheral T-cell lymphomas. Thus, this double staining provided the authors with valuable information regarding the proportion, localization, and surface phenotype of proliferating cells, which should be useful for diagnosis of the diseases of lymphoid system.
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PMID:Double immunoenzymatic detection of surface phenotype of proliferating lymphocytes in situ with monoclonal antibodies against DNA polymerase alpha and lymphocyte membrane antigens. 243 27

Human DNA polymerase alpha holoenzyme follows an ordered sequential terreactant mechanism of substrate recognition and binding (Wong, S. W., Paborsky, L. R., Fisher, P. A., Wang, T. S.-F., and Korn, D. (1986) J. Biol. Chem. 261, 7958-7968). We confirmed this mechanism for the DNA polymerase alpha holoenzyme purified from Drosophila melanogaster embryos and studied the interaction of Drosophila pol alpha with synthetic oligonucleotide template-primers containing modified tetrahydrofuran moieties as model abasic lesions chemically engineered at a number of defined sites. Abasic lesions in the template had relatively little effect on the polymerase incorporation reaction at sites proximal to the lesion. However, incorporation opposite an abasic site was undetectable relative to that which occurred opposite a normal template nucleotide. Moreover, abasic residues in the primer region of the template-primer construct as far as 4 base pairs removed from the 3'-primer terminus prevented detectable nucleotide incorporation relative to that seen on an unmodified template-primer. Primer-region lesions had qualitatively similar effects whether they were located on the primer strand itself or on the complementary template strand. Data from polymerase incorporation experiments were corroborated by competitive binding assays performed under steady state reaction conditions. Results of these experiments suggested that polymerase binding to synthetic oligonucleotide template-primers was essentially unaffected by lesions located at sites that did not block incorporation. Lesions that did block incorporation apparently did so by abrogating template-primer binding. These observations have implications for understanding the mechanisms whereby DNA polymerase alpha recognizes noninformational template sites in vivo and prevents DNA synthesis from proceeding past these points.
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PMID:Recognition and binding of template-primers containing defined abasic sites by Drosophila DNA polymerase alpha holoenzyme. 250 45

A flow cytometric method to analyze phenotypes of proliferative cells was developed using human leukemic cell line MOLT 4. A nuclear protein, DNA polymerase alpha (pol alpha), was selected as a marker for proliferative cells, and Leu3a molecule as a cell-surface antigen phenotype marker of the cells. The procedure involved the simultaneous use of fluorescein-conjugated anti-pol alpha antibody, developed by us, and commercially available phycoerythrin-conjugated anti-Leu3a antibody. The optimal fixative for both proteins was phosphate-buffered 2% paraformaldehyde. The pol alpha-positive population in logarythmically growing MOLT 4 cells was estimated, by flow cytometry, to be ca. 95%. A sharp flow cytometry histogram with a strong pol alpha-linked fluorescence was observed. On the other hand, the pol alpha-positive population in the saturated culture was ca. 70%, with weaker pol alpha-linked fluorescence. Thus, the population of pol alpha-positive cells and the amount of pol alpha in cells was dependent on the cell density of the culture. In contrast, ca. 90% Leu3a-positive populations with similar flow cytometry histograms were seen in either growing or saturated states, suggesting that expression of Leu3a was independent of cell density. The flow cytometric method using fluorescein isothiocyanate-conjugated anti-pol alpha antibody is useful for detecting proliferative fractions of free tumor cells, such as leukemic cells. Furthermore, analysis of the phenotype of the proliferative or non-proliferative cells became easier by simultaneous labeling with antibodies against pol alpha and phenotype-specific proteins.
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PMID:Expression of DNA polymerase alpha and Leu3a molecules in growing and saturated cultures of human leukemic cells: phenotype analysis of proliferative cells by flow cytometry. 251 70

Seven herpes simplex virus (HSV) genes have been shown recently to be necessary and sufficient to support the replication of origin-containing plasmids. Two of these genes (pol and dbp) encode well-known DNA replication proteins (the DNA polymerase and the major single-stranded DNA binding protein), and a third gene (UL42) encodes a previously identified infected-cell protein which binds tightly to double-stranded DNA. The products of the four remaining genes have not previously been identified. Using the predicted amino acid sequence data (D.J. McGeoch, M.A. Dalrymple, A. Dolan, D. McNab, L.J. Perry, P. Taylor, and M.D. Challberg, J. Virol. 62:444-453; D.J. McGeoch and J.P. Quinn, Nucleic Acids Res. 13:8143-8163), we have raised rabbit antisera against the products of all seven genes. We report here the use of these reagents to identify these proteins in infected cells. All seven proteins localized to the nucleus and were expressed in a manner consistent with the idea that they are the products of early genes. Various immunological assays suggest that four of these proteins (UL5, UL8, UL9, and UL52) are made in infected cells in very low abundance relative to the other three. To improve our ability to study these proteins, we have expressed UL5, UL8, UL9, and UL52 in insect cells by using the baculovirus expression system. The HSV protein made in insect cells were immunoprecipitable with the appropriate antisera, and the size of each protein was indistinguishable from the size of the corresponding protein made in HSV-infected Vero cells. Our data offer strong support for the accuracy of open reading frames proposed by McGeoch et al. In addition, the antisera and the overproduced HSV replication proteins should be useful reagents with which to analyze the biochemistry of HSV DNA replication.
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PMID:Herpes simplex virus type 1 gene products required for DNA replication: identification and overexpression. 253 26

The 65-kilodalton DNA-binding protein (65KDBP) of herpes simplex virus type 1 (HSV-1), the product of the UL42 gene, is required for DNA replication both in vitro and in vivo, yet its actual function is unknown. By two independent methods, it was shown that the 65KDBP stimulates the activity of the HSV-1-encoded DNA polymerase (Pol). When Pol, purified from HSV-1-infected cells, was separated from the 65KDBP, much of its activity was lost. However, addition of the 65KDBP, purified from infected cells, stimulated the activity of Pol 4- to 10-fold. The ability of a monoclonal antibody to the 65KDBP to remove the Pol-stimulating activity from preparations of the 65KDBP confirmed that the activity was not due to a trace contaminant. Furthermore, the 65KDBP did not stimulate the activity of other DNA polymerases derived from T4, T7, or Escherichia coli. The 65KDBP gene transcribed in vitro from cloned DNA and translated in vitro in rabbit reticulocyte lysates also was capable of stimulating the product of the pol gene when the RNAs were cotranslated. The product of a mutant 65KDBP gene missing the carboxy-terminal 28 amino acids exhibited wild-type levels of Pol stimulation, while the products of two large deletion mutants of the gene could not stimulate Pol activity. These experiments suggest that the 65KDBP may be an accessory protein for the HSV-1 Pol.
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PMID:The essential 65-kilodalton DNA-binding protein of herpes simplex virus stimulates the virus-encoded DNA polymerase. 255 39


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