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PubMed1975

Specificity of the antiviral agent calcium elenolate.

Calcium elenolate, an antiviral agent which inhibits reverse transcriptases, inhibits the growth of chicken embryo fibroblast cells, as well as Echerichia coli and Bacillus subtilis strains. The drug in vitro inhibits E. coli deoxyribonucleic acid (DNA) polymerase II and DNA polymerase III holoenzyme, as well as several unrelated enzymes. The usual DNA polymerase assay components, with the exception of spermidine, have no effect on the observed inhibition. Inhibition of DNA polymerase II by the drug appears to be due to a direct and irreversible effect on the enzyme. However, DNA synthesis in E. coli is no more susceptible to the drug than is the increase in cell mass. These results suggest that calcium elenolate is an inhibitor of rather low specificity.

Animals↗
PubMed2005

Roles of the Escherichia coli RecA protein and the global SOS response in effecting DNA polymerase selection in vivo.

The Escherichia coli beta sliding clamp protein is proposed to play an important role in effecting switches between different DNA polymerases during replication, repair, and translesion DNA synthesis. We recently described how strains bearing the dnaN159 allele, which encodes a mutant form of the beta clamp (beta159), display a UV-sensitive phenotype that is suppressed by inactivation of DNA polymerase IV (M. D. Sutton, J. Bacteriol. 186:6738-6748, 2004). As part of an ongoing effort to understand mechanisms of DNA polymerase management in E. coli, we have further characterized effects of the dnaN159 allele on polymerase usage. Three of the five E.coli DNA polymerases (II, IV, and V) are regulated as part of the global SOS response. Our results indicate that elevated expression of the dinB-encoded polymerase IV is sufficient to result in conditional lethality of the dnaN159 strain. In contrast, chronically activated RecA protein, expressed from the recA730 allele, is lethal to the dnaN159 strain, and this lethality is suppressed by mutations that either mitigate RecA730 activity (i.e., DeltarecR), or impair the activities of DNA polymerase II or DNA polymerase V (i.e., DeltapolB or DeltaumuDC). Thus, we have identified distinct genetic requirements whereby each of the three different SOS-regulated DNA polymerases are able to confer lethality upon the dnaN159 strain, suggesting the presence of multiple mechanisms by which the actions of the cell's different DNA polymerases are managed in vivo.

DNA Polymerase III↗
PubMed1999

Analysis of unassisted translesion replication by the DNA polymerase III holoenzyme.

DNA damage-induced mutations are formed when damaged nucleotides present in single-stranded DNA are replicated. We have developed a new method for the preparation of gapped plasmids containing site-specific damaged nucleotides, as model DNA substrates for translesion replication. Using these substrates, we show that the DNA polymerase III holoenzyme from Escherichia coli can bypass a synthetic abasic site analogue with high efficiency (30% bypass in 16 min), unassisted by other proteins. The theta and tau subunits of the polymerase were not essential for bypass. No bypass was observed when the enzyme was assayed on a synthetic 60-mer oligonucleotide carrying the same lesion, and bypass on a linear gapped plasmid was 3-4-fold slower than on a circular gapped plasmid. There was no difference in the bypass when standing-start and running-start replication were compared. A comparison of translesion replication by DNA polymerase I, DNA polymerase II, the DNA polymerase III core, and the DNA polymerase III holoenzyme clearly showed that the DNA polymerase III holoenzyme was by far the most effective in performing translesion replication. This was not only due to the high processivity of the pol III holoenzyme, because increasing the processivity of pol II by adding the gamma complex and beta subunit, did not increase bypass. These results support the model that SOS regulation was imposed on a fundamentally constitutive translesion replication reaction to achieve tight control of mutagenesis.

Base Composition↗
PubMed1999

The kinetoplast structure-specific endonuclease I is related to the 5' exo/endonuclease domain of bacterial DNA polymerase I and colocalizes with the kinetoplast topoisomerase II and DNA polymerase beta during replication.

The mitochondrial DNA (kinetoplast DNA) of the trypanosomatid Crithidia fasciculata has an unusual structure composed of minicircles and maxicircles topologically interlocked into a single network and organized in a disc-shaped structure at the base of the flagellum. We previously purified a structure-specific endonuclease (SSE1), based on its RNase H activity, that is enriched in isolated kinetoplasts. The endonuclease gene has now been cloned, sequenced, and found to be closely related to the 5' exonuclease domain of bacterial DNA polymerase I proteins. Although the protein does not contain a typical mitochondrial leader sequence, the enzyme is shown to colocalize with a type II DNA topoisomerase and a DNA polymerase beta at antipodal sites flanking the kinetoplast disc. Cell synchronization studies with an epitope-tagged construct show that the localization of the endonuclease to the antipodal sites varies in a cell cycle-dependent manner similar to that of the DNA polymerase beta [Johnson, C. E. & Englund, P. T. (1998) J. Cell Biol. 143, 911-919]. Immunofluorescent localization of SSE1 to the antipodal sites is only observed during kinetoplast replication. Together, these results suggest a point of control for kinetoplast DNA replication through the regulation of the availability of DNA replication proteins and a possible role for the antipodal sites in removal of RNA primers and the repair of gaps in newly replicated minicircles.

Amino Acid Sequence↗
PubMed1976

Template properties of human DNA-dependent RNA polymerase II.

DNA-dependent RNA polymerase II has been purified from lymphocytes of patients with chronic lymphocytic leukemia (CLL). Form II polymerase and Escherichia coli RNA polymerase have been used to study the transcription of human DNA and chromatin. The hybridization kinetics of the transcripts of DNA and chromatin by the human polymerase are quite different; chromatin transcripts hybridized to DNA at a much slower rate than DNA transcripts, whereas the transcripts of DNA and chromatin by bacterial polymerase have similar hybridization kinetics.

Centrifugation, Density Gradient↗
PubMed1982

Effects of exogenous polyamine and trypanocides on the DNA polymerase activities from Trypanosoma brucei brucei, mouse thymus and murine leukemia virus.

The effects of exogenously added spermine on activated (gapped) DNA-directed and poly(dC) . (dG)12-18-directed DNA synthesis were tested on the chromatographically separated DNA polymerase activities of Trypanosoma brucei brucei. Activated DNA-directed DNA synthesis by the Peak I (eluting from DNA-agarose at 0.15 M KCl) and Peak II (eluting at 0.3 M KCl) polymerase was consistently inhibited or stimulated, respectively, by exogenous spermine. Kinetic analysis revealed that inhibition of the Peak I enzyme with respect to template DNA occurred by a mixed mechanism, while a major factor in the stimulation of the Peak II enzyme by spermine appeared to be the polyamine-mediated reversal of "substrate inhibition' by DNA at concentrations above 10 micrograms/ml. The apparent Km values of Peak I and Peak II DNA polymerase for activated DNA were determined to be 5 and 0.5 microgram/ml, respectively. In contrast to the results observed with activated DNA, activation of Peak II-enzyme-catalyzed poly(dC)-directed DNA synthesis was similar at all template-primer concentrations. Peak I enzyme-catalyzed poly(dG) synthesis was either inhibited or slightly stimulated by spermine, depending upon the presence or absence of heteropolymeric DNA, respectively. Dose-dependent inhibition of DNA-directed DNA synthesis catalyzed by T. b. brucei DNA polymerases, murine thymus DNA polymerase alpha, and Rauscher murine leukemia virus reverse transcriptase by trypanocides was examined to determine a possible mechanism of selective toxicity by such agents. The drugs Antrycide (quinapyramine), pentamidine, imidocarb, Berenil (diminazene aceturate), WR-199-385-[2,5-bis(4-guanylphenyl)furan . 2HCl] and isometamidium inhibited DNA polymerases of the eucaryotic cells at approximately the same degree, and at similar concentrations. The presence of spermine in reaction mixtures did not spare any drug inhibition. Stimulation of reverse transcriptase activity was observed in the presence of Antrycide and imidocarb, however, this could be negated by stimulatory amounts of spermine present in the reaction mixture. The results, obtained using an activated DNA-directed assay system, suggest that trypanosomal DNA polymerases are not the selective target of trypanocidal drugs currently available.

Animals↗
PubMed1977

Purification using polyethylenimine precipitation and low molecular weight subunit analyses of calf thymus and wheat germ DNA-dependent RNA polymerase II.

DNA-dependent RNA polymerase II from calf thymus has been successfully purified using polythylenimine precipitation. Thus, 5-6 mg of nearly homogeneous homogeneous trna polymerase II (greater than 96% pure) can be prepared from 1 kg of calf thymus with three chromatography steps following extraction and precipitation of the enzyme from the polyethylenimine pellet. This procedure eliminates the high salt extraction of chromatin previously used in purification of this enzyme and makes possible the large scale preparation of mammalian RNA polymerase II. Calf thymus polymerase II prepared by this method is greater than 90% form IIb and consists of ten different subunits having the following molecular weights: 180 000; 145 000; 36 000; 25 000; 20 000; 18 500; 16 000; 15 000; 12 000; 11 500. The homologous enzyme isolated from wheat germ is greater than 90% form IIa and contains subunits of the following molecular weights: 206 000; 145 000; 44 000-47 000; 24 500; 21 000; 19 000; 17 000; 14 000; 13 500. The wheat germ and calf thymus enzymes exhibit similar subunits structures, but the molecular weights of individual subunits are clearly different between the enzymes. Wheat germ RNA polymerase II is 50% inhibited by 0.271 microng/mL of alpha-amanitin, a level 30-fold higher than that found for calf thymus RNA polymerase II. These enzymes are further distinguished by the absence of antigenic cross reactivity.

Animals↗
PubMed2002

Apoptosis: biochemical aspects and clinical implications.

Apoptosis and necrosis represent two distinct types of cell death. Apoptosis possesses unique morphologic and biochemical features which distinguish this mechanism of programmed cell death from necrosis. Extrinsic apoptotic cell death is receptor-linked and initiates apoptosis by activating caspase 8. Intrinsic apoptotic cell death is mediated by the release of cytochrome c from mitochondrial and initiates apoptosis by activating caspase 3. Cancer chemotherapy utilizes apoptosis to eliminate tumor cells. Agents which bind to the minor groove of DNA, like camptothecin and Hoechst 33342, inhibit topoisomerase I, RNA polymerase II, DNA polymerase and initiate intrinsic apoptotic cell death. Hoechst 33342-induced apoptosis is associated with disruption of TATA box binding protein/TATA box complexes, replication protein A/single-stranded DNA complexes, topoisomerase I/DNA cleavable complexes and with an increased intracellular concentration of E2F-1 transcription factor and nitric oxide concentration. Nitric oxide and transcription factor activation or respression also regulate the two apoptotic pathways. Some human diseases are associated with excess or deficient rates of apoptosis, and therapeutic strategies to regulate the rate of apoptosis include inhibition or activation of caspases, mRNA antisense to reduce anti-apoptotic factors like Bcl-2 and survivin and recombinant TRAIL to activate pro-apoptotic receptors, DR4 and DR5.

Animals↗
PubMed1974

DNA repair in Escherichia coli mutants deficient in DNA polymerases I, II and-or 3.

E. coli mutants deficient in DNA polymerase I, in DNA polymerases I and II, or in DNA polymerase III, can efficiently and completely execute excision repair and post-replication repair of UV-damaged DNA at 43 degrees when assayed by alkaline sucrose gradients. Repair by cells deficient in polymerase I and in polymerases I and II is inhibited by 1-beta-D-arabinofuranosylcytosine at 43 degrees , whereas that by cells deficient in polymerase III is insensitive to the inhibitor. When both DNA polymerases I and III are deficient, both excision repair and post-replication repair are greatly reduced at 43 degrees , and the residual repair capability is inhibited by 1-beta-D-arabinofuranosylcytosine. Very little dark repair is observed in cells deficient in DNA polymerases I, II, and III, and the DNA is extensively degraded. These results suggest that either DNA polymerase I or DNA polymerase III is required for complete and efficient repair, and that when both DNA polymerases I and III are deficient, DNA polymerase II mediates a limited, incomplete dark repair of UV-damaged DNA. DNA polymerases I and III thus appear to be important enzymes in both DNA replication and DNA dark repair.

Carbon Radioisotopes↗
PubMed2005

Evidence for Escherichia coli polymerase II mutagenic bypass of intrastrand DNA crosslinks.

The mutagenic potentials of DNAs containing site- and stereospecific intrastrand DNA crosslinks were evaluated in Escherichia coli cells that contained a full complement of DNA polymerases or were deficient in either polymerases II, IV, or V. Crosslinks were made between adjacent N(6)-N(6) adenines and consisted of R,R- and S,S-butadiene crosslinks and unfunctionalized 2-, 3-, and 4-carbon tethers. Although replication of single-stranded DNAs containing the unfunctionalized 3- and 4-carbon tethers were non-mutagenic in all strains tested, replication past all the other intrastrand crosslinks was mutagenic in all E. coli strains, except the one deficient in polymerase II in which no mutations were ever detected. However, when mutagenesis was analyzed in cells induced for SOS, mutations were not detected, suggesting a possible change in the overall fidelity of polymerase II under SOS conditions. These data suggest that DNA polymerase II is responsible for the in vivo mutagenic bypass of these lesions in wild-type E. coli.

DNA↗
PubMed1991

RNase-like domain in DNA-directed RNA polymerase II.

DNA-directed RNA polymerase is responsible for gene expression. Despite its importance, many details of its function and higher-order structure still remain unknown. We report here a local sequence similarity between the second largest subunit of RNA polymerase II and bacterial RNases Ba (barnase), Bi, and St. The most remarkable similarity is that the catalytic sites of the RNases are shared with the eukaryotic RNA polymerase II subunits of Drosophila melanogaster and Saccharomyces cerevisiae. Several amino acids conserved among the RNases and the RNase-like domains of the RNA polymerase subunits are located in the neighborhood of the catalytic sites of barnase, whose three-dimensional structure has been resolved. This observation suggests the functional importance of the RNase-like domain of the RNA polymerase subunits and indicates that the RNase-like domain may have RNase activity. The location of the RNase-like domain relative to the region necessary for RNA polymerization is similar to the relative proximity of 5'----3' or 3'----5' exonuclease and the region of polymerase activity of DNA polymerase I. The RNase-like domain might work in proofreading, as in RNA-directed RNA polymerase of influenza virus, or it may contribute to RNA binding through an unknown function.

Amino Acid Sequence↗
PubMed1994

Molecular modeling of RNA polymerase II mutations onto DNA polymerase I.

Genetic and molecular analysis in Drosophila melanogaster identifies eight suppressor mutations in the second largest subunit of RNA polymerase II. The suppressor mutations fall into two classes: five are strong, result from the same serine to cysteine amino acid residue substitution and rescue one conditional lethal allele in the largest subunit of RNA polymerase II; three are mild, result from a change in the same methionine residue to either isoleucine or valine, are located seven amino acid residues away from the strong suppressors and rescue two conditional lethal alleles in the largest subunit. Sequence analysis of the three regions around these mutations demonstrates that they are located within highly conserved domains but fails to explain the observed genetic interactions. One of the conditional lethal alleles maps within a region previously reported to share sequence similarity to Escherichia coli DNA polymerase I. As the gross structure of RNA polymerase II and DNA polymerase I is similar, even though their primary sequence is not, we predict that more similarities exist but may be too highly divergent to be detected by normal homology searches. We identify the most similar regions between each of the three conserved domains of RNA polymerase II, identified as functionally important because of the mutations we isolated, and DNA polymerase I. Molecular modeling these regions of RNA polymerase II onto the tertiary structure of DNA polymerase I predicts that all lie adjacent to the DNA binding cleft in positions such that they could interact with the phosphate backbone of DNA. This juxtaposition of mutations in the two largest subunits of RNA polymerase II suggest a mechanism for their genetic interactions.

Amino Acid Sequence↗
PubMed1994

2-Chloro-2'-deoxyadenosine monophosphate residues in DNA enhance susceptibility to 3'-->5' exonucleases.

2-Chloro-2'-deoxyadenosine triphosphate, a purine nucleotide analogue and potent antileukaemic agent, was incorporated into double-stranded 36-mers in place of dATP to investigate the effects of 2-chloroadenine (ClAde) on DNA polymerase-associated 3'-->5' exonuclease activity. ClAde residues within one strand of duplex DNA did not inhibit exonuclease activity; on the contrary, ClAde-containing minus strands were digested to a greater extent than was control DNA in the absence of deoxyribonucleoside triphosphates by Escherichia coli Klenow fragment, yeast DNA polymerase II and T4 DNA polymerase. After a 30 min incubation with 5 units of Klenow fragment, approximately 65% of control DNA remained in DNA fragments of 26 bases or larger compared with only approximately 25% of ClAde-substituted substrates. Unsubstituted plus strands opposite a ClAde-containing strand were likewise digested more quickly by 3'-->5' exonuclease, but only in the vicinity of the ClAde sites. Approx. 63% of the plus strands from ClAde-containing oligomers were less than 24 bases in length after a 25 min digestion period with Klenow fragment compared with only approximately 32% of control DNA. Such results indicate that, unlike other base modifications such as pyrimidine dimers, methoxy psoralen adducts and certain nucleoside analogues, all of which inhibit or decrease the rate of strand degradation by 3'-->5' exonucleases, incorporated ClAde enhances strand degradation of duplex DNA.

Adenine↗
PubMed1998

A novel DNA polymerase family found in Archaea.

One of the most puzzling results from the complete genome sequence of the methanogenic archaeon Methanococcus jannaschii was that the organism may have only one DNA polymerase gene. This is because no other DNA polymerase-like open reading frames (ORFs) were found besides one ORF having the typical alpha-like DNA polymerase (family B). Recently, we identified the genes of DNA polymerase II (the second DNA polymerase) from the hyperthermophilic archaeon Pyrococcus furiosus, which has also at least one alpha-like DNA polymerase (T. Uemori, Y. Sato, I. Kato, H. Doi, and Y. Ishino, Genes Cells 2:499-512, 1997). The genes in M. jannaschii encoding the proteins that are homologous to the DNA polymerase II of P. furiosus have been located and cloned. The gene products of M. jannaschii expressed in Escherichia coli had both DNA polymerizing and 3'-->5' exonuclease activities. We propose here a novel DNA polymerase family which is entirely different from other hitherto-described DNA polymerases.

Amino Acid Sequence↗
PubMed1976

Mechanism of DNA elongation catalyzed by Escherichia coli DNA polymerase III, dnaZ protein, and DNA elongation factors I and III.

Elongation of a primed single-stranded DNA template catalyzed by E. coli DNA polymerase III (DNA nucleotidyltransferase, deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase, EC 2.7.7.7) requires dnaZ protein and two other protein factors, DNA elongation factors I and III. The reaction occurs by the following mechanism: (i) dnaZ protein and DNA elongation factor III together catalyze the transfer of DNA elongation factor I to a primed DNA template. This transfer reaction requires ATP or dATP in addition to dnaZ protein, DNA elongation factors I and III, and primed template; it does not require DNA polymerase III. (ii) DNA polymerase III binds to the complex of DNA elongation factor I with primed template; it does not bind to primed template which is not complexed with DNA elongation factor I. This binding reaction proceeds in the absence of ATP or dATP as cofactor, dnaZ protein, and DNA elongation factor III and without additional DNA elongation factor I. (iii) The complex of DNA polymerase III, DNA elongation factor I, and primed template catalyzes DNA synthesis upon the addition of dNTPs.

Adenosine Triphosphate↗
PubMed1991

Replication factors required for SV40 DNA replication in vitro. II. Switching of DNA polymerase alpha and delta during initiation of leading and lagging strand synthesis.

Replication factors A and C (RF-A and RF-C) and the proliferating cell nuclear antigen (PCNA) differentially augment the activities of DNA polymerases alpha and delta. The mechanism of stimulation by these replication factors was investigated using a limiting concentration of primed, single-stranded template DNA. RF-A stimulated polymerase alpha activity in a concentration-dependent manner, but also suppressed nonspecific initiation of DNA synthesis by both polymerases alpha and delta. The primer recognition complex, RF-C.PCNA.ATP, stimulated pol delta activity in cooperation with RF-A, but also functioned to prevent abnormal initiation of DNA synthesis by polymerase alpha. Reconstitution of DNA replication with purified factors and a plasmid containing the SV40 origin sequences directly demonstrated DNA polymerase alpha dependent synthesis of lagging strands and DNA polymerase delta/PCNA/RF-C dependent synthesis of leading strands. RF-A and the primer recognition complex both affected the relative levels of leading and lagging strands. These results, in addition to results in an accompanying paper (Tsurimoto, T., and Stillman, B. (1991) J. Biol. Chem. 266, 1950-1960), suggest that an exchange of DNA polymerase complexes occurs during initiation of bidirectional DNA replication at the SV40 origin.

Adenosine Triphosphate↗
PubMed1985

Chinese hamster ovary cell mutants resistant to DNA polymerase inhibitors. II. Segregational analysis and DNA transfection of the aphr gene.

Genetic characterizations of the Chinese hamster ovary cell mutants resistant to the DNA polymerase inhibitors (aphidicolin, ara-A and ara-C) have been described. Resistance to all three inhibitors showed dominance among the progeny of somatic cell crosses between the wild type and mutant parents. Analysis of the segregation of the drug-resistant character among 566 hybrid progeny from somatic crosses between the wild type (aphs, ara-As, and ara-Cs) and the triple mutants (aphr, ara-Ar, ara-Cr) showed the involvement of at least three unlinked genes in controlling the expression of the resistance to different DNA polymerase inhibitors. The mutant (aphr) DNA was used to transfect aphidicolin resistance to recipient mouse Ltk- cells either directly or in combination with the plasmid pTK2 DNA. The aphidicolin resistance of the transfected cells was found to be a stable phenotype and could be used in multiple rounds of transfection, indicating the chromosomal integration of the transfecting gene.

Animals↗