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)

Poly(L-malate) is an unusual polyanion found in nuclei of plasmodia of Physarum polycephalum. We have investigated, by enzymatic and fluorimetric methods, whether poly(L-malate) and structurally related polyanions can interact with DNA-polymerase-alpha-primase complex and with histones of P. polycephalum. Poly(L-malate) is found to inhibit the activities of the DNA-polymerase-alpha-primase complex and to bind to histones. The mode of inhibition is competitive with regard to DNA in elongation and noncompetitive in the priming of DNA synthesis. Spermidine, spermine, and histones from P. polycephalum and from calf thymus bind to poly(L-malate) and antagonize the inhibition. The polyanions poly(vinyl sulfate), poly(acrylate), poly(L-malate), poly(D,L-malate), poly(L-aspartate), poly(L-glutamate) have been examined for their potency to inhibit the DNA polymerase. The degree of inhibition is found to depend on the distance between neighboring charges, given by the number of atoms (N) interspaced between them. Poly(L-malate) (N = 5) and poly(D,L-malate) (N = 5) are the most efficient inhibitors, followed by poly(L-aspartate) (N = 6), poly(acrylate) (N = 3), poly(L-glutamate) (N = 8), poly(vinyl sulfate) (N = 3). It is proposed that poly(L-malate) interacts with DNA-polymerase-alpha-primase of P. polycephalum. According to its physical and biochemical properties, poly(L-malate) may alternatively function as a molecular chaperone in nucleosome assembly in the S phase and as both an inhibitor and a stock-piling agent of DNA-polymerase-alpha-primase in the G2 phase and M phase of the plasmodial cell cycle.
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PMID:Specific inhibition of Physarum polycephalum DNA-polymerase-alpha-primase by poly(L-malate) and related polyanions. 137 54

DNA polymerase II purified from Saccharomyces cerevisiae contains polypeptides with apparent molecular masses of greater than 200, 80, 34, 30 and 29 kDa, the two largest of which (subunits A and B) are encoded by the essential genes POL2 and DPB2. By probing a lambda gt11 expression library of yeast DNA with antiserum against DNA polymerase II, we isolated a single gene, DPB3, that encodes both the 34- and 30-kDa polypeptides (subunit C and C'). The nucleotide sequence of DPB3 contained an open reading frame encoding a 23-kDa protein, significantly smaller than the observed molecular masses, 34- or 30-kDa, which might represent post-translationally modified forms of the DPB3 product. The predicted amino acid sequence contained a possible NTP-binding motif and a glutamate-rich region. NTP-binding motif and a glutamate-rich region. A dpb3 deletion mutant (dpb3 delta) was viable and yielded a DNA polymerase II lacking the 34- and 30-kDa polypeptides. dpb3 delta strains exhibited an increased spontaneous mutation rate, suggesting that the DPB3 product is required to maintain fidelity of chromosomal replication. Since a fifth, 29-kDa polypeptide was present in DNA polymerase II preparations from wild-type cell extracts throughout purification, the subunit composition appears to be A, B, C (or C and C') and D. The 5' nontranscribed region of DPB3 contained the MulI-related sequence ACGCGA, while the 0.9-kb DPB3 transcript accumulated periodically during the cell cycle and peaked at the G1/S boundary. The level of DPB3 transcript thus appears to be under the same cell cycle control as those of POL2, DPB2 and other DNA replication genes. DPB3 was mapped to chromosome II, 30 cM distal to his7.
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PMID:Cloning DPB3, the gene encoding the third subunit of DNA polymerase II of Saccharomyces cerevisiae. 192 54

The beta subunit of DNA polymerase III holoenzyme is in a dimer-monomer equilibrium at physiological beta concentrations. Dissociation is accompanied by the fluorescence enhancement of a fluorophore attached to a unique sulfhydryl group of beta (Griep, M. A., and McHenry, C. S. (1988) Biochemistry 27, 5210-5215). Sequencing of the isolated tryptic peptides of beta revealed that the fluorescent maleimide group was attached to cysteine 333. The 2 residues, lysine 332 and glutamate 334, that flank this residue are hydrophilic and may place cysteine 333 on the surface of beta, explaining its high reactivity. Fluorescence energy transfer permitted us to locate the uniquely labeled cysteines 333 of beta at the distal ends of the beta dimer. When the beta dimer was dissociated to monomers, the accompanying alteration of the conformational state was reported by the fluorescein-5-maleimide (fluorescein)-labeled cysteines which were located far from the dimer interface. The carboxyl of fluorescein had a fluorescence pKa of 6.9 when beta was in its dimeric state. The pKa decreased by 0.3 pH unit upon dissociation to monomers and resulted in the fluorescence enhancement that was observed when the signal was monitored at constant pH. The adjacent glutamate 334 apparently increased the pKa of the attached fluorescein when beta was in its dimeric state. Movement of either the adjacent lysine 332 amino side chain to a closer position or glutamate 334 to a position further away could lower the pKa upon beta monomerization. Thus, beta undergoes a conformational change concomitant with dimer dissociation that was transmitted to the opposite ends of the beta dimer. The pKa of fluorescein attached to the distal cysteines was shifted, leading to greater ionization and enhanced fluorescence.
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PMID:Dissociation of the DNA polymerase III holoenzyme beta 2 subunits is accompanied by conformational change at distal cysteines 333. 224 96

Even though Escherichia coli can grow in media containing up to 1 M NaCl, one-fifth that amount of NaCl will completely inhibit the in vitro activity of DNA polymerase III holoenzyme. It has been established that the major intracellular ionic osmolytes are potassium and glutamate (Richey, B., Cayley, D. S., Mossing, M. C., Kolka, C., Anderson, C. F., Farrar, T. C., and Record, M. T., Jr. (1987) J. Biol. Chem. 262, 7157-7164). We have found that holoenzyme catalyzes replication efficiently in vitro in up to 1 M potassium glutamate. Two salt effects on the replication of single-stranded DNA were observed. At low salt replicative activity was enhanced and at high salt there was anion-specific inhibition. We have found that DNA polymerase III holoenzyme tolerated 10-fold higher concentrations of glutamate than chloride. The ability of various anions to extend the useful range of salt concentrations followed the order: phosphate less than chloride less than N-Ac-glutamate less than acetate less than glycine less than aspartate less than glutamate. With the exception of phosphate, this order followed the Hofmeister series indicating that the anion-specific effects were due to anions interacting at the protein-water interface at weak anion binding sites. Glutamate did not reverse the inhibition by chloride. The low salt enhancement and high salt inhibition effects were additive for the two anions indicating that they competed for common anion binding sites. The major salt-sensitive step was holoenzyme binding to template rather than the subsequent elongation reaction.
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PMID:Glutamate overcomes the salt inhibition of DNA polymerase III holoenzyme. 256 34

The presence and the level of hepatitis B surface antigen (HBsAg)/IgM complexes were determined in 54 chronic HBsAg carriers in relation to receptors for polymerized human serum albumin (pHSA-R) tested by specific radioimmunoassay, and to hepatitis B virus-DNA polymerase (HBV-DNAp). HBsAg/IgM complexes, correlated significantly with the HBsAg concentration but, at a similar HBsAg concentration, significant highest values of HBsAg/IgM complexes were found among HBeAg positive patients. In addition, a significant correlation was found between HBsAg/IgM complex levels, HBeAg titres and HBV-DNAp activity (r = 0.628, p less than 0.001 and r = 0.559, p less than 0.001, respectively). Moreover, a positive linear correlation was found when comparing HBsAg/IgM complexes and pHSA-R levels (r = 0.848, p less than 0.001). Patients who were positive for HBsAg/IgM complexes had a significantly higher glutamate-pyruvate transaminase (GPT) level than those who did not show any complexes. In conclusion, HBsAg/IgM complexes seemed to be indirectly related to HBV replication.
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PMID:Hepatitis B surface antigen/IgM complexes: relation to receptors for polymerized human serum albumin, hepatitis B virus (HBV) DNA polymerase activity and HBV markers. 288 20

The effects of ticlopidine and six of its analogues on mitochondrial functions were studied in isolated rat liver mitochondria. The influence of ticlopidine and each of the following analogues: PCR 5325, PCR 4099, PCR 3787, PCR 2362, PCR 4499 and PCR 0665 was evaluated by determining their interaction with three major mitochondrial activities. (A) Oxidative phosphorylation, measured by oxypolarography, was assayed in the presence of glutamate or succinate as source of energy, and both State 4 and State 3 were recorded. Ticlopidine, at 20 micrograms/ml, slightly increased glutamate State 4, whereas it was without effect on that of succinate. At higher concentration (40 micrograms/ml), ticlopidine caused 40-45% inhibition of State 4 with both substrates. All the other analogues tested at either 20 or 40 micrograms/ml were virtually without effect on the respiration. However, at 20 micrograms/ml, ticlopidine and some of its analogues inhibited mitochondrial State 3, while under similar conditions other analogues had little or no effect on this state. (B) Mitochondrial protein synthesis, measured by [14C]-L-leucine incorporation, was not affected significantly by any of these drugs. Whereas chloramphenicol at 10 micrograms/ml caused 80% inhibition, ticlopidine and its analogues in concentrations inhibitory to State 3 did not inhibit mitochondrial protein synthesis. (C) Mitochondrial DNA polymerase activity, determined by [3H] thymidine 5'-triphosphate incorporation, was not inhibited by these drugs. We conclude that, while ticlopidine and analogues have little or no effect on either mitochondrial protein synthesis or mitochondrial DNA polymerase activity, ticlopidine and some of its analogues are inhibitory of the energy conserving mechanism in mitochondria.
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PMID:Effects of ticlopidine, a new platelet antiaggregating agent, and its analogues on mitochondrial metabolism. Oxidative phosphorylation, protein synthesis and DNA polymerase activity. 650 40

DNA polymerase epsilon (pol epsilon) from HeLa cells was purified to near homogeneity, utilizing Mono S fast protein liquid chromatography for complete separation from pol alpha. The purified pol epsilon preparation showed two polypeptides of > 200 and 55 kDa and a small amount of active 122-kDa proteolysis product on denaturing polyacrylamide gels. Pol epsilon (as well as pols alpha and delta) is optimally active in 100-150 mM potassium glutamate and 15 mM MgCl2. Replication factors RF-A and RF-C, proliferating cell nuclear antigen, and Escherichia coli single-stranded DNA binding protein showed no significant effect on this preparation's pol epsilon activity, processivity, or substrate specificity. The size of the pol epsilon transcript for the catalytic subunit (> 200 kDa) was investigated in both normal human fibroblasts and HeLa cells. A 7.7-kilobase transcript was detected which was 5-16-fold more prevalent in proliferating than in quiescent HeLa cells. No significant difference in the level of pol epsilon transcript in HeLa cells or fibroblasts was seen after ultraviolet irradiation. Mouse polyclonal antiserum was produced to a 144-amino acid fragment of pol epsilon fused to staphylococcal protein A. This non-neutralizing polyclonal antiserum specifically recognized the catalytic subunit of pol epsilon by immunoblotting, but not that of pol alpha, beta, or delta. In addition, mouse polyclonal antiserum raised against column-purified pol epsilon was able to recognize and to neutralize pol epsilon, and a mouse monoclonal antibody was raised which was able to recognize specifically the catalytic subunit of pol epsilon.
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PMID:Further characterization of HeLa DNA polymerase epsilon. 753 91

Ten mutator alleles of MIP1, the gene encoding mitochondrial (mt) DNA polymerase, have been isolated after in vitro random mutagenesis. Five mutations causing a 100-400-fold increase in the frequency of erythromycin-resistant (ErR) mt mutants in yeast mapped to the 3'-5' exonuclease (Exo) domain, and mainly to the three conserved motifs Exo1, Exo2 and Exo3 of this domain, highlighting the importance of proofreading in accurate mt DNA replication. The essential role of the invariant glutamate at the Exo1 site was confirmed and the participation of four amino acids (aa) in the 3'-5' Exo function revealed. Another mutation that is located between the Exo1 and Exo2 sites produced an extremely strong mutator phenotype associated with impaired DNA replication, but could be assigned neither to a conserved aa nor to a conserved portion of the 3'-5' exonuclease domain. The importance of the polymerization domain in accurate mt DNA replication was pointed out by three mutator mutations. Two of these severely impaired mt DNA replication and were assigned to a subdomain of the polymerase which probably corresponds to the 'fingers' module of the Klenow (large) fragment of Escherichia coli DNA polymerase I (PolIk). The third, which did not alter the efficiency of DNA replication, was located at the active center of the polymerization reaction. Finally, the mutation, R1001I, mapped to the C-terminal part of the MIP1 protein which has no counterpart in prokaryotic DNA polymerases.
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PMID:Isolation and characterization of ten mutator alleles of the mitochondrial DNA polymerase-encoding MIP1 gene from Saccharomyces cerevisiae. 762 2

Endonuclease digestion of the 4,800-kb chromosome of Salmonella typhimurium LT2 yielded 24 XbaI fragments, 12 BlnI fragments, and 7 CeuI fragments, which were separated by pulsed-field gel electrophoresis. The 90-kb plasmid pSLT has one XbaI site and one BlnI site. The locations of the fragments around the circular chromosome and of the digestion sites of the different endonucleases with respect to each other were determined by excision of agarose blocks containing fragments from single digestion, redigestion with a second enzyme, end labelling with 32P by using T7 DNA polymerase, reelectrophoresis, and autoradiography. Forty-three cleavage sites were established on the chromosome, and the fragments and cleavage sites were designated in alphabetical order and numerical order, respectively, around the chromosome. One hundred nine independent Tn10 insertions previously mapped by genetic means were located by pulsed-field gel electrophoresis on the basis of the presence of XbaI and BlnI sites in Tn10. The genomic cleavage map was divided into 100 units called centisomes; the endonuclease cleavage sites and the genes defined by the positions of Tn10 insertions were located by centisome around the map. There is very good agreement between the genomic cleavage map, defined in centisomes, and the linkage map, defined in minutes. All seven rRNA genes were located on the map; all have the CeuI digestion site, all four with the tRNA gene for glutamate have the XbaI and the BlnI sites, but only four of the seven have the BlnI site in the 16S rRNA (rrs) gene. Their inferred orientation of transcription is the same as in Escherichia coli. A rearrangement of the rrnB and rrnD genes with respect to the arrangement in E. coli, observed earlier by others, has been confirmed. The sites for all three enzymes in the rrn genes are strongly conserved compared with those in E. coli, but the XbaI and BlnI sites outside the rrn genes show very little conservation.
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PMID:The XbaI-BlnI-CeuI genomic cleavage map of Salmonella typhimurium LT2 determined by double digestion, end labelling, and pulsed-field gel electrophoresis. 832 Feb 26

The replicative polymerase of Escherichia coli, DNA polymerase III, consists of a three-subunit core polymerase plus seven accessory subunits. Of these seven, tau and gamma are products of one replication gene, dnaX. The shorter gamma is created from within the tau reading frame by a programmed ribosomal -1 frameshift over codons 428 and 429 followed by a stop codon in the new frame. Two temperature-sensitive mutations are available in dnaX. The 2016(Ts) mutation altered both tau and gamma by changing codon 118 from glycine to aspartate; the 36(Ts) mutation affected the activity only of tau because it altered codon 601 (from glutamate to lysine). Evidence which indicates that, of these two proteins, only the longer tau is essential includes the following. (i) The 36(Ts) mutation is a temperature-sensitive lethal allele, and overproduction of wild-type gamma cannot restore its growth. (ii) An allele which produced tau only could be substituted for the wild-type chromosomal gene, but a gamma-only allele could not substitute for the wild-type dnaX in the haploid state. Thus, the shorter subunit gamma is not essential, suggesting that tau can be substitute for the usual function(s) of gamma. Consistent with these results, we found that a functional polymerase was assembled from nine pure subunits in the absence of the gamma subunit. However, the possibility that, in cells growing without gamma, proteolysis of tau to form a gamma-like product in amounts below the Western blot (immunoblot) sensitivity level cannot be excluded.
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PMID:The Escherichia coli DNA polymerase III holoenzyme contains both products of the dnaX gene, tau and gamma, but only tau is essential. 837 47


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