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)

A mini-Tn10 insertion in the polA cistron (polA2099) was isolated in a search for mutations that affect patterned Mudlac replication in colonies. The polA2099 mutation had a dramatic effect on cell morphogenesis during the first few hours of microcolony development. Abnormal microcolonies containing filamentous cells were produced as a result of SOS induction. Despite gross abnormalities in early microcolonies, mature polA2099 colonies after 2 to 4 days were morphologically indistinguishable from Pol+ colonies, and 44-h polA2099 colonies displayed a cell size distribution very similar to that of Pol+ colonies. These results suggested the involvement of a protective factor produced during colony growth that compensated for the polA deficiency. The action of a diffusible substance that stimulates growth of polA2099 microcolonies was shown by spotting dilute polA2099 cultures next to established colonies. Differential transcription of polA during colony development was visualized by growing colonies containing polA-lacZ fusions on beta-galactosidase indicator agar. When polA-lacZ colonies were inoculated next to established colonies, a diffusible factor was seen to inhibit polA transcription during the earliest stages of colony development. These results show that a basic housekeeping function, DNA polymerase I, is subject to multicellular control by the changing conditions which the bacteria create as they proliferate on agar.
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PMID:Differential action and differential expression of DNA polymerase I during Escherichia coli colony development. 133 Oct 25

Screening of a Mycobacterium tuberculosis genomic DNA library in the lambda gt11 expression vector was carried out by using, as probes, sera from tuberculous patients and murine monoclonal antibody H61.3 recognizing a mycobacterial 35-kilodalton protein present only on the M. tuberculosis complex. The recombinant beta-galactosidase-fused protein present in the crude lysate induced the proliferation of T lymphocytes from patients with tuberculous pleuritis. As the recombinant insert contains an internal EcoRI restriction site, it was possible to identify two fragments, one proximal to the lacZ gene and 1.7 kilobases (kb) in size and the other distal to the lacZ gene and 2.2 kb in size. Southern blot analysis showed that both of them hybridized with the genomic DNA from M. tuberculosis and M. bovis but not with the DNA from other mycobacterial species. To perform extensive immunological studies, the amount of beta-galactosidase-fused protein being very low, we fused the 1.7-kb fragment to the N-terminal part of the gene coding for the DNA polymerase of bacteriophage MS2 in the expression vector pEx34. The fusion protein was partially purified, and subsequent Western blotting (immunoblotting) and T-cell proliferation experiments confirmed the presence of B- and T-cell mycobacterial epitopes. Furthermore, to isolate the chromosomal region containing the 35-kilodalton gene, we constructed another mycobacterial genomic library in the lambda 2001 vector by cloning 15 to 20 kb of foreign DNA. Screening of this library was carried out by using 1.7- and 2.2-kb recombinant fragments as probes. Restriction maps of some clones isolated were determined.
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PMID:Identification of a 35-kilodalton Mycobacterium tuberculosis protein containing B- and T-cell epitopes. 168 20

Adenovirus DNA polymerase (AdPol) contains three clusters of basic amino acids within the N-terminal 48 amino acids: RARR, which begins at amino acid 8, RRRVR, which begins at amino acid 25, and RARRRR, which begins at amino acid 41. These clusters are designated BS I, BS II, and BS III, respectively. (The amino acid codes are: R, arginine; A, alanine; V, valine.) Mutational analysis of these noncontiguous clusters showed that AdPol contains a novel organization of bipartite nuclear localization signals (NLS) that interact differentially to serve in the nuclear targeting of AdPol or of chimeric proteins in which AdPol is linked to Escherichia coli beta-galactosidase (beta-gal). The region containing BS I and BS II functioned interdependently as an NLS for the nuclear targeting of AdPol, for which BS III was dispensible. However, the region containing BS II and BS III constituted a second and more efficient bipartite NLS for the nuclear targeting of the AdPol-E. coli beta-gal fusion protein. Moreover, deletion or limited insertion of amino acids in the spacer region between BS II and BS III did not affect their nuclear targeting function for these fusion proteins. Chou-Fasman predictive analysis of protein secondary structure in the vicinity of the bipartite NLS sequences supports a model in which protein conformation in the spacer region may play an important role in bringing these clusters of basic amino acids into close proximity, allowing them to function as nuclear targeting signals for this class of nuclear proteins.
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PMID:Three basic regions in adenovirus DNA polymerase interact differentially depending on the protein context to function as bipartite nuclear localization signals. 177 81

Many of the proteins that operate at the replication fork in Escherichia coli have been defined genetically. These include some of the subunits of the DNA polymerase III holoenzyme, the DnaB replication fork helicase, and the DnaG primase. The multiprotein primosome (which includes the DnaB and DnaG proteins), defined biochemically on the basis of its requirement during bacteriophage phi X174 complementary-strand synthesis, could serve as the helicase-primase replication machine on the lagging-strand template. In order to determine if this is the case, we have begun an investigation of the phenotypes of mutants with mutations priA, priB, and priC, which encode the primosomal proteins factor Y (protein n'), n, and n", respectively. Inactivation of priA by insertional mutagenesis resulted in the induction of the SOS response, as evinced by induction of a resident lambda prophage, extreme filamentation, and derepression of an indicator operon in which beta-galactosidase production was controlled by the dinD1 promoter. In addition, the copy numbers of resident pBR322 plasmids were reduced four- to fivefold in these strains, and production of phi X174 phage was delayed considerably. These results are discussed in the context of existing models for SOS induction and possible roles for the PriA protein at the replication fork in vivo.
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PMID:Inactivation of the Escherichia coli priA DNA replication protein induces the SOS response. 193 75

We purified a mouse DNA repair enzyme having apurinic/apyrimidinic endonuclease, DNA 3'-phosphatase, 3'-5'-exonuclease and DNA 3' repair diesterase activities, and designated the enzyme as APEX nuclease. A cDNA clone for the enzyme was isolated from a mouse spleen cDNA library using probes of degenerate oligonucleotides deduced from the N-terminal amino acid sequence of the enzyme. The complete nucleotide sequence of the cDNA (1.3 kilobases) was determined. Northern hybridization using this cDNA showed that the size of its mRNA is about 1.5 kilobases. The complete amino acid sequence for the enzyme predicted from the nucleotide sequence of the cDNA (APEX nuclease cDNA) indicates that the enzyme consists of 316 amino acids with a calculated molecular weight of 35,400. The predicted sequence contains the partial amino acid sequences determined by a protein sequencer from the purified enzyme. The coding sequence of APEX nuclease was cloned into pUC18 SmaI and HindIII sites in the control frame of the lacZ promoter. The construct was introduced into BW2001 (xth-11, nfo-2) strain cells of Escherichia coli. The transformed cells expressed a 36.4-kDa polypeptide (the 316 amino acid sequence of APEX nuclease headed by the N-terminal decapeptide of beta-galactosidase) and were less sensitive to methyl methanesulfonate than the parent cells. The fusion product showed priming activity for DNA polymerase on bleomycin-damaged DNA and acid-depurinated DNA. The deduced amino acid sequence of mouse APEX nuclease exhibits a significant homology to those of exonuclease III of E. coli and ExoA protein of Streptococcus pneumoniae and an intensive homology with that of bovine AP endonuclease 1.
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PMID:cDNA and deduced amino acid sequence of a mouse DNA repair enzyme (APEX nuclease) with significant homology to Escherichia coli exonuclease III. 193 31

The Schizosaccharomyces pombe POL3 gene was isolated by sequence homology with a region of the Saccharomyces cerevisiae POL3 gene, the only gene sequenced to date encoding the catalytic subunit of eukaryotic DNA polymerase delta. The fission yeast POL3 gene contains a 52 base-pair (bp) intron and encodes a 3600 bp transcript the 5'-end of which is located 32 bp upstream from the initiation codon. The polypeptides predicted from budding and fission yeast POL3 genes share 52% of conserved amino acid residues and have a 60% identical central region. This structural conservation of the catalytic subunit of DNA polymerases delta is probably related to functional constraints. A portion of the most conserved region was used to raise antibodies against an S. pombe polymerase delta/beta-galactosidase fusion protein expressed in Escherichia coli. The purified antibodies recognized a 123,000 Da protein in S. pombe wild-type cell extracts and inhibited an aphidicolin-sensitive DNA polymerase activity that was distinct from DNA polymerase alpha. The antibodies also detected a 140,000 Da protein in extracts from different proliferating mammalian cells, indicating that the catalytic subunits of DNA polymerase delta are highly conserved between yeast and higher eukaryotes.
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PMID:Characterization of the POL3 gene product from Schizosaccharomyces pombe indicates inter-species conservation of the catalytic subunit of DNA polymerase delta. 196 Jul 23

Spot 42 RNA of Escherichia coli, a 109-nucleotide RNA that influences the level of DNA polymerase I, has an AUG triplet preceded by a purine-rich potential ribosome-binding site and is followed by a short (14-triplet) potential open reading frame. Although the RNA bound to ribosomes, it did so inefficiently and nonproductively. When fused to lacZ sequences, spot RNA did not support the synthesis of beta-galactosidase. Also, the biological effects of spot 42 RNA were not altered by mutation of the tyrosine UAU codon to the chain termination UAG. We conclude that the effects of spot 42 RNA are mediated by the RNA itself and not by a spot 42 RNA-encoded peptide.
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PMID:Spot 42 RNA of Escherichia coli is not an mRNA. 244 Aug 52

Sequence studies of the adenovirus 2 genome have revealed the presence of a large open reading frame (ORF) from 22.9 to 14.2 map units that is believed to encode most of the adenovirus DNA polymerase (Ad Pol). An 838-base-pair fragment (19.6-17.3 map units) containing approximately 25% of this ORF has been cloned and expressed in a beta-galactosidase-chloramphenicol acetyltransferase (lacZ-CAT) expression vector under the control of the trp-lac hybrid promoter. This recombinant vector directed the synthesis of a 58-kDa lacZ-Ad Pol-CAT fusion protein that has CAT activity. This fusion protein was easily purified by affinity chromatography in which chloramphenicol, the substrate for CAT, was covalently bound to a matrix. Antisera were prepared against the purified 58-kDa lacZ-Ad Pol-CAT fusion protein and were found to react specifically with the 140-kDa Ad Pol by ELISA and immunoblot analysis. In addition, these antisera recognized 120- and 29-kDa polypeptides in immunoblot analysis of partially purified terminal protein precursor (pTP)-Ad Pol complex. The exact nature of the 120- and 29-kDa polypeptides is not known, but they may be breakdown products of Ad Pol. Although the lacZ-Ad Pol-CAT fusion protein is not active in any of the Ad Pol enzymatic reactions, antibody against the prokaryotic fusion protein should be useful for screening bacteria harboring plasmids that have been constructed to express the entire Ad Pol ORF.
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PMID:The 140-kDa adenovirus DNA polymerase is recognized by antibodies to Escherichia coli-synthesized determinants predicted from an open reading frame on the adenovirus genome. 258 Dec 53

A 1.4 kb region downstream of the DNA polymerase gene of Autographa californica nuclear polyhedrosis virus was sequenced. Two open reading frames (ORFs) were identified of 927 and 474 bases in length. The 927 base ORF encodes a 34.8K protein as determined by in vitro translation of both hybrid-selected RNA and RNA synthesized in vitro from a 927 base ORF template. The predicted amino acid sequence of the 34.8K polypeptide (p34.8) reveals a hydrophobic N terminus, two potential N-glycosylation sites, and potential sites for phosphorylation by casein kinase I and protein kinase C. The p34.8 gene has a strong codon usage bias which is strikingly different from that of the polyhedrin gene. The two 5' ends of the 927 base ORF transcripts initiate from an ATAAG sequence and a GTAAG sequence 11 and 87 bases upstream of the ATG codon respectively. A short upstream reading frame is present in the leader sequence of the longer RNA. The transcripts have multiple 3' ends; the most proximal endpoint correlates with a polyadenylation signal overlapping the translational termination codon of the 927 base ORF. Transcripts of the latter were not observed early in the infection cycle but appeared 6 h after infection and were maximally expressed at 12 to 24 h post-infection. The late nature of these transcripts was confirmed by their sensitivity to aphidicolin and cycloheximide, inhibitors of DNA replication and protein synthesis respectively. Attempts to construct viral mutants carrying a deletion of the p34.8 gene and fusion with the beta-galactosidase gene suggest that the former gene is essential for viral replication.
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PMID:Sequence, transcription and translation of a late gene of the Autographa californica nuclear polyhedrosis virus encoding a 34.8K polypeptide. 267 27

The dnaZX gene of Escherichia coli directs the synthesis of two proteins, DnaZ and DnaX. These products are confirmed as the gamma and tau subunits of DNA polymerase III because antibody to a synthetic peptide present in both the DnaZ and DnaX proteins reacts also with the gamma and tau subunits of holoenzyme. To characterize biochemically the tau subunit, for which there has been no activity assay, the dnaZX gene was fused to the beta-galactosidase gene to encode a fusion product in which the 20 C-terminal amino acids of the DnaX protein (tau) were replaced by beta-galactosidase lacking only 7 N-terminal amino acids. The 185-kDa fusion protein, which retained beta-galactosidase activity, was overproduced to the level of about 5% of the soluble cellular protein by placing the gene fusion under control of the tac promoter and Shine-Dalgarno sequence. The fusion protein was isolated in one step by affinity chromatography on p-aminobenzyl 1-thio-beta-D-galactopyranoside-agarose. The purified fusion protein also had ATPase (and dATPase) activity that was dependent on single-stranded DNA. This activity copurified with the beta-galactosidase activity not only through the affinity column but also through a subsequent gel filtration. We conclude that the DnaX protein function involves binding to single-stranded DNA and hydrolysis of ATP or dATP, in addition to binding to other DNA polymerase III holoenzyme components, increasing the processivity of the core enzyme, and serving as a substrate for the production of the gamma subunit.
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PMID:Escherichia coli DnaX product, the tau subunit of DNA polymerase III, is a multifunctional protein with single-stranded DNA-dependent ATPase activity. 303 60


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