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)

Helix-hairpin-helix (HhH) is a widespread motif involved in sequence-nonspecific DNA binding. The majority of HhH motifs function as DNA-binding modules with typical occurrence of one HhH motif or one or two (HhH)(2) domains in proteins. We recently identified 24 HhH motifs in DNA topoisomerase V (Topo V). Although these motifs are dispensable for the topoisomerase activity of Topo V, their removal narrows the salt concentration range for topoisomerase activity tenfold. Here, we demonstrate the utility of Topo V's HhH motifs for modulating DNA-binding properties of the Stoffel fragment of TaqDNA polymerase and Pfu DNA polymerase. Different HhH cassettes fused with either NH(2) terminus or COOH terminus of DNA polymerases broaden the salt concentration range of the polymerase activity significantly (up to 0.5 M NaCl or 1.8 M potassium glutamate). We found that anions play a major role in the inhibition of DNA polymerase activity. The resistance of initial extension rates and the processivity of chimeric polymerases to salts depend on the structure of added HhH motifs. Regardless of the type of the construct, the thermal stability of chimeric Taq polymerases increases under the optimal ionic conditions, as compared with that of TaqDNA polymerase or its Stoffel fragment. Our approach to raise the salt tolerance, processivity, and thermostability of Taq and Pfu DNA polymerases may be applied to all pol1- and polB-type polymerases, as well as to other DNA processing enzymes.
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PMID:Helix-hairpin-helix motifs confer salt resistance and processivity on chimeric DNA polymerases. 1236 75

The beta subunit of Escherichia coli DNA polymerase III holoenzyme was fused to the green fluorescent protein GFP. The gene fusion under the control of the heterologous lac promoter was used to replace the wild-type allele in the chromosome. The formation of GFP-beta fluorescent foci in GFP-beta expressing cells required DNA replication and their number per cell was dependent on cell growth. Examination of GFP-beta foci in a synchronous round of replication suggested that DNA replication was accompanied by the recruitment of GFP-beta foci near the midcell, followed by the rapid migration of the foci in opposite directions to the 1/4 and 3/4 positions during DNA replication.
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PMID:Cellular localisation of the clamp protein during DNA replication. 1243 11

Proper segregation of chromosomes during mitosis requires that the products of chromosome replication are paired together-termed sister chromatid cohesion. In budding yeast, Ctf7p/Eco1p is an essential protein that establishes cohesion between sister chromatids during S phase. In fission yeast, Eso1p also functions in cohesion establishment, but is comprised of a Ctf7p/Eco1p domain fused to a Rad30p domain (a DNA polymerase) both of which are independently expressed in budding yeast. In this report, we identify and characterize the first candidate human ortholog of Ctf7p/Eco1p, which we term hEFO1p (human Establishment Factor Ortholog). As in fission yeast Eso1p, the hEFO1p open reading frame extends well upstream of the C-terminal Ctf7p/Eco1p domain. However, this N-terminal extension in hEFO1p is unlike Rad30p, but instead exhibits significant homology to linker histone proteins. Thus, hEFO1p is a unique fusion of linker histone and cohesion establishment domains. hEFO1p is widely expressed among the tissues tested. Consistent with a role in chromosome segregation, hEFO1p localizes exclusively to the nucleus when expressed in HeLa tissue culture cells. Moreover, biochemical analyses reveal that hEFO1p exhibits acetyltransferase activity. These findings document the first characterization of a novel human acetyltransferase, hEFO1p, that is comprised of both linker histone and Ctf7p/Eco1p domains.
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PMID:Human EFO1p exhibits acetyltransferase activity and is a unique combination of linker histone and Ctf7p/Eco1p chromatid cohesion establishment domains. 1457 21

Nucleocytoplasmic trafficking of functional proteins plays a key role in regulating gene expressions in response to extracellular signals. We developed a genetically encoded bioluminescent indicator for monitoring the nuclear trafficking of target proteins in vitro and in vivo. The principle is based on reconstitution of split fragments of Renilla reniformis (Rluc) by protein splicing with a DnaE intein (a catalytic subunit of DNA polymerase III). A target cytosolic protein fused to the N-terminal half of Rluc is expressed in mammalian cells. If the protein translocates into the nucleus, the Rluc moiety meets the C-terminal half of Rluc, and full-length Rluc is reconstituted by protein splicing. We demonstrated quantitative cell-based in vitro sensing of ligand-induced translocation of androgen receptor, which allowed high-throughput screening of exo- and endogenous agonists and antagonists. Furthermore, the indicator enabled noninvasive in vivo imaging of the androgen receptor translocation in the brains of living mice with a charge-coupled device imaging system. These rapid and quantitative analyses in vitro and in vivo provide a wide variety of applications for screening pharmacological or toxicological compounds and testing them in living animals.
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PMID:High-throughput sensing and noninvasive imaging of protein nuclear transport by using reconstitution of split Renilla luciferase. 1528 15

The REV1 gene encodes a Y-family DNA polymerase that has been postulated to have both catalytic and structural functions in translesion replication past UV photoproducts in mammalian cells. To examine if REV1 is implicated in DNA damage tolerance mechanisms after exposure of human cells to a chemical carcinogen, we generated a plasmid expressing REV1 protein fused at its C-terminus with green fluorescent protein (GFP). In transient transfection experiments, virtually all of the transfected cells had a diffuse nuclear pattern in the absence of carcinogen exposure. In contrast, in cells exposed to benzo[a]pyrenediolepoxide, the fusion protein accumulated in a focal pattern in the nucleus in 25% of the cells, and co-localized with PCNA. These data support the idea that REV1 is present at stalled replication forks. We also examined the mutagenic response at the HPRT locus of human cells that had greatly reduced levels of REV1 mRNA due to the stable expression of gene-specific ribozymes, and compared them to wild-type cells. The mutant frequency was greatly reduced in the ribozyme-expressing cells. These data indicate that REV1 is implicated in the mutagenic DNA damage tolerance response to BPDE and support the development of strategies to target this protein to prevent such mutations.
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PMID:REV1 accumulates in DNA damage-induced nuclear foci in human cells and is implicated in mutagenesis by benzo[a]pyrenediolepoxide. 1552 96

The infected-cell protein 22 (ICP22), a regulatory protein encoded by the alpha22 gene of herpes simplex virus 1, is required for the optimal expression of a set of late viral proteins that includes the products of the U(S)11, U(L)38, and U(L)41 genes. ICP22 has two activities. Thus, ICP22 and the U(L)13 protein kinase mediate the activation of cdc2 and degradation of its partners, cyclins A and B. cdc2 and its new partner, the DNA polymerase accessory factor (U(L)42), bind topoisomerase IIalpha in an ICP22-dependent manner. In addition, ICP22 and U(L)13 mediate an intermediate phosphorylation of the carboxyl terminus of RNA polymerase II (RNA POL II). Here we report another function of ICP22. Thus, ICP22 physically interacts with cdk9, a constitutively active cyclin-dependent kinase involved in transcriptional regulation. A protein complex containing ICP22 and cdk9 phosphorylates in vitro the carboxyl-terminal domain of RNA POL II in a viral U(S)3 protein kinase-dependent fashion. Finally, the carboxyl-terminal domain of RNA POL II fused to glutathione S-transferase is phosphorylated in reaction mixtures containing complexes pulled down with ICP22 or cdk9 immune precipitated from lysates of wild-type parent virus or deltaU(L)13 but not deltaU(S)3 mutant-infected cells. The experiments described here place ICP22 and cdk9 in a complex with the carboxyl-terminal domain of RNA POL II. At the same time we confirm the requirement of ICP22 and the U(L)13 protein kinase in the posttranslational modification of RNA POL II that alters its electrophoretic mobility, although U(S)3 kinase appears to play a role in a cell-type-dependent fashion.
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PMID:The carboxyl-terminal domain of RNA polymerase II is phosphorylated by a complex containing cdk9 and infected-cell protein 22 of herpes simplex virus 1. 1589 Sep 14

Based on our previous experience with arylsulfone derivatives displaying antiherpetic activity, we synthesized several analogues in which the sulfonyl group is part of a bicyclic structure. The benzene-fused derivative 2H-3-(4-chlorophenyl)-3,4-dihydro-1,4-benzo-thiazine-2-carbonitrile 1,1-dioxide and its thiophene-fused analogue were shown to have favorable activity and selectivity against the betaherpesviruses human cytomegalovirus (HCMV) and human herpesvirus 6 (HHV-6) and 7 (HHV-7). The benzene-fused derivative retained its anti-HCMV activity when evaluated against virus strains resistant to foscarnet, ganciclovir, and/or cidofovir. The compound conferred >or=95% inhibition of viral DNA synthesis in HHV-6-infected cells. RT-PCR analysis of immediate-early, early and late gene products revealed that this arylsulfone compound acts at a step preceding late gene expression, and coinciding with the inhibition exerted by foscarnet. No inhibitory effect was seen in an enzyme assay for DNA elongation catalyzed by the HCMV or HHV-6 DNA polymerase catalytic subunit. The arylsulfone derivatives had no effect on the functional interaction between the catalytic subunit of HCMV DNA polymerase and its accessory protein, nor did they disrupt the physical interaction between the two proteins. We conclude that these arylsulfone derivatives represent new betaherpesvirus inhibitors with a novel mode of action that results in indirect inhibition of viral DNA synthesis.
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PMID:Antiviral properties of new arylsulfone derivatives with activity against human betaherpesviruses. 1665 Apr 89

In this study, we report that the partitioning between mitochondria and cytoplasm of two variants, mCherry and DsRed Express (DRE), of the red fluorescent protein, DsRed, fused to one of the six matrix targeting sequences (MTSs) can be affected by both MTS and amino acid substitutions in DsRed. Of the six MTSs tested, MTSs from superoxide dismutase and DNA polymerase gamma failed to direct mCherry, but not DRE to mitochondria. By evaluating a series of chimeras between mCherry and DRE fused to the MTS of superoxide dismutase, we attribute the differences in the mitochondrial partitioning to differences in the primary amino acid sequence of the passenger polypeptide. The impairment of mitochondrial partitioning closely parallels the number of mCherry-specific mutations, and is not specific to mutations located in any particular region of the polypeptide. These observations suggest that both MTS and the passenger polypeptide affect the efficiency of mitochondrial import and provide a rationale for the observed diversity in the primary amino acid sequences of natural MTSs.
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PMID:Mutations in the passenger polypeptide can affect its partitioning between mitochondria and cytoplasm: mutations can impair the mitochondrial import of DsRed. 1738 58

Herpes simplex virus 1 (HSV-1) strain ANG and ANGpath were cloned as bacterial artificial chromosome (BAC). Two different types of BAC genomes were obtained. BAC genomes of type I contained the BAC replicon at the intended target region between the genes of UL48 and UL49. In BAC genomes of type II, the BAC sequences were found to be aberrantly fused between the termini of the HSV-1 genome. Both the BAC types were used to establish a conditional gene expression system for HSV-1 by Flp recombinase-mediated insertion of expression vectors that were modified to respond to the T-REx tetracycline (Tet)-inducible transcription switch. During BAC cloning and mutagenesis in E. coli, not only deletions but also defined mutations of the HSV-1 genome were observed. Successful virus reconstitution from BACs with large inserts demonstrated that HSV-1 has a packaging capacity for foreign sequences of at least 8.1% of its genome size. Targets for Tet-regulated gene expression were the viral DNA polymerase gene (pol) and a reporter gene of glycoprotein B fused to enhanced green fluorescent protein (gBGFP). Results with the pol gene as target showed that virus plaque production could not be significantly controlled by the T-REx gene switch using vectors encoding one copy of the tetR gene. In contrast, an efficient Tet-response was achieved with the gBGFP reporter, which was optimal in a Tet repressor (TetR)-producing cell line, demonstrating that the TetR concentration provided by the virus was not sufficient for a tight control of Tet-regulated gene expression.
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PMID:Evaluation of the T-REx transcription switch for conditional expression and regulation of HSV-1 vectors. 1819 47

Optical nanostructures have enabled the creation of subdiffraction detection volumes for single-molecule fluorescence microscopy. Their applicability is extended by the ability to place molecules in the confined observation volume without interfering with their biological function. Here, we demonstrate that processive DNA synthesis thousands of bases in length was carried out by individual DNA polymerase molecules immobilized in the observation volumes of zero-mode waveguides (ZMWs) in high-density arrays. Selective immobilization of polymerase to the fused silica floor of the ZMW was achieved by passivation of the metal cladding surface using polyphosphonate chemistry, producing enzyme density contrasts of glass over aluminum in excess of 400:1. Yields of single-molecule occupancies of approximately 30% were obtained for a range of ZMW diameters (70-100 nm). Results presented here support the application of immobilized single DNA polymerases in ZMW arrays for long-read-length DNA sequencing.
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PMID:Selective aluminum passivation for targeted immobilization of single DNA polymerase molecules in zero-mode waveguide nanostructures. 1821 53


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