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Biomedical subjects

L A Loeb

Publications and source records attributed to L A Loeb.

At least 235 records · Page 13Linked to original sources
PubMed1978

Apparent suicidal inactivation of DNA polymerase by adenosine 2',3'-riboepoxide 5'-triphosphate.

Adenosine 2',3'-riboepoxide 5'-triphosphate (epoxyATP) has been found to be a suicidal inactivator of DNA polymerase I from Escherichia coli by the following criteria. Inactivation is complete, is first order in enzyme activity, and shows saturation kinetics with an apparent KD of 30 +/- 10 micron for epoxy ATP. This KD is comparable to the KM of the substrate dATP. The t1/2 for inactivation is 1.3 min. Inactivation requires Mg2+ and the complementary template. The enzyme is protected by dATP but not by an excess of template. Gel filtration of the reaction mixture after inactivation with [3H]epoxy ATP results in the comigration of E. coli DNA polymerase I, the tritium-labeled inactivator, and the DNA template. The stoichiometry of binding approaches 1 mol of [3H]epoxy nucleotide per mol of inactivated enzyme. These results are consistent with the hypothesis that epoxy ATP initially serves as a substrate for the polymerase reaction, elongating the DNA chain by a nucleotidyl unit, and subsequently alkylates an essential base at the primer terminus binding site of the enzyme. Epoxy ATP also inactivates human and viral DNA polymerases but not E. coli RNA polymerase or rabbit muscle pyruvate kinase. Hence epoxy ATP may be a specific suicide reagent for DNA polymerases.

Adenosine Triphosphate↗
PubMed1978

DNA replication in human lymphocytes during aging.

An analysis of the accuracy of protein and DNA synthesis in human lymphocytes with respect to aging has been carried out. The response of human peripheral lymphocytes, from young and old adults, to phytohemagglutinin was measured at varying temperatures. This should provide a sensitive test for the accumulation of altered thermolabile proteins that are rate limiting in the response to phytohemagglutinin. At 37 degrees C the rate of thymidine incorporation as well as the induction of DNA polymerase in phytohemagglutinin-stimulated lymphocytes from old and young adults were similar. Also at elevated temperatures, the thermosensitivity of DNA replication in lymphocytes from young and old adults was the same. DNA polymerase was purified from PHA-stimulated lymphocytes from young and old adults. The fidelity of DNA synthesis using poly (dC) as a template was similar with both enzymes. However, DNA polymerase-alpha purified from old adults was thermolabile compared to the enzyme from young adults. Thus, while the lymphocytes from old individuals may have heat labile proteins, they do not limit their proliferative capacity.

Adult↗
PubMed1978

Mutagenesis during in vitro DNA synthesis.

The error frequency of in vitro DNA synthesis using a natural DNA template has been measured with a biological assay for nucleotide substitutions. phiX174 DNA containing an amber mutation was copied in vitro by Escherichia coli DNA polymerase I, and the reversion frequency of the progeny DNA was determined by transfection of E. coli spheroplasts. E. coli polymerase I makes less than 1 mistake at the am3 locus for every 7700 nucleotides incorporated under standard reaction conditions. Substitution of Mn2+ for Mg2+ and unequal concentrations of deoxynucleoside triphosphate substrates raises this mutation frequency to greater than 1 in 1000. Thus, E. coli DNA polymerase I can copy natural DNA templates with high fidelity and its accuracy can be affected by alterations in reaction conditions.

Coliphages↗
PubMed1977

On the fidelity of DNA replication. Effect of metal activators during synthesis with avian myeloblastosis virus DNA polymerase.

The effect of metal activators on the fidelity of DNA synthesis has been examined. Using the DNA polymerase from avian myeloblastosis virus, the accuracy of Co2+-, M2+-, and Ni2+-activated DNA synthesis was determined with different polynucleotide templates. With poly[d(A-T)] as the template, the error frequency for dCMP incorporation was 1:1400, 1:1100, and 1:600 for Mg2+, Co2+, and Mn2+, respectively, at maximally activating concentrations. The error frequency was invariant with respect to [Mg2+] but increased with greater than activating concentrations of Co2+ and Mn2+. This increase resulted from differential rates of complementary and noncomplementary nucleotide incorporation. The enhanced error frequency was nonspecific as it occurred with all polynucleotide templates and with all noncomplementary deoxy- and ribonucleotides which were tested. Nearest neighbor analyses of the reaction products indicated that the noncomplementary deoxynucleotides were incorporated as single base substitutions. The fidelity of Ni2+-activated DNA synthesis was invariant with respect to [Ni2+] and was similar to that obtained using Mg2+. During DNA synthesis with Mg2+, the addition of Co2+, Mn2+, or Ni2+ resulted in a decrease in the fidelity of DNA synthesis. The relationship between decreases in the fidelity of DNA synthesis and metal mutagenesis, or carcinogenesis, or both, is considered.

Avian Myeloblastosis Virus↗
PubMed1977

Infidelity of DNA synthesis as related to mutagenesis and carcinogenesis.

An assay system has been developed for measuring the fidelity of DNA synthesis in vitro by using synthetic polynucleotide templates and purified DNA polymerases. Nearest-neighbor analysis of the synthesized product indicates that noncomplementary nucleotides are incorporated as single base substitutions. The accuracy of DNA synthesis can be decreased by (1) prior alkylation of the template, (2) increasing the relative concentration of incorrect nucleotides, and (3) addition of specific metal salts to the reaction mixture. As an initial evaluation of the utility of this system, the effects of 31 metal salts on the fidelity of DNA synthesis have been determined. The results indicate that potential metal mutagens and/or carcinogens may be detected by measuring alterations in the fidelity of DNA synthesis.

Avian Myeloblastosis Virus↗
PubMed1977

Screening for deficits in DNA repair by the response of irradiated human lymphocytes to phytohemagglutinin.

An assay has been developed to measure the ability of human lymphocytes to repair damage to DNA. In this assay, purified human lymphocytes are exposed to graded doses of radiation and then stimulated with phytohemagglutinin to undergo DNA replication. The rate of incorporation of thymidine in irradiated lymphocytes during the second and subsequent rounds of DNA replication is taken to be indicative of the ability of the cells to repair damage to DNA. In lymphocytes from normal individuals, X-irradiation with doses of 100 to 800 rads was found to inhibit phytohemagglutinin-stimulated thymidine incorporation proportionally to the dose of radiation without curtailing the induction of DNA polymerase. The response to phytohemagglutinin of lymphocytes from a patient with xeroderma pigmentosum after exposure to graded doses of X-irradiation was found to be similar to that of the normal controls, whereas the response after ultraviolet irradiation was markedly impaired. In contrast, lymphocytes from patients with ataxia telangiectasia were hypersensitive to X-irradiation. The data on these clinical syndromes support the idea that this assay measures DNA repair and indicates the feasibility of using this method for screening individuals for genetic deficits in DNA repair.

Ataxia Telangiectasia↗
PubMed1976

Infidelity of DNA synthesis in vitro: screening for potential metal mutagens or carcinogens.

Thirty-one metal salts have been tested for their ability to affect the accuracy of DNA synthesis in vitro. All ten salts of metal carcinogens decreased the fidelity of DNA synthesis. Of the three metals which beforehand were considered to be possible mutagens or carcinogens, only one decreased fidelity. In contrast, 17 noncarcinogenic metal salts did not affect fidelity even when present at concentrations that were clearly inhibitory.

Carcinogens↗
PubMed1976

On the association of reverse transcriptase with polynucleotide templates during catalysis.

The association of avian myeloblastosis virus (AMV) DNA polymerase with polynucleotide templates during catalysis has been studied. During the course of polymerization, different template-primer complexes were added and the ability of the enzyme to switch from one polynucleotide template to another was determined. At 37 degrees C as well as at 4 degrees C, the polymerase is able to switch from certain template-primer complexes to others. For example, the addition of poly(A)-oligo(dT) during the course of synthesis with poly(C)-oligo(dG) results in the immediate cessation of dGMP polymerization and the start of dTMP polymerization without any lag. Early during the course of polymerization, the size of the product, as determined by alkaline sucrose gradient centrifugation, is, in part, a function of the ratio of the template-primer complex to the enzyme. These cumulative experiments indicate that catalysis on polynucleotide templates with avian myeloblastosis virus DNA polymerase under the conditions tested is not processive in a classical sense. Similar to cellular DNA polymerases the enzyme can shift from one template-primer to another. Using autoradiography after gel electrophoresis to estimate the product size, it can be calculated that the enzyme switches from one template to another within 0.25 min at 37 degrees C which corresponds to the incorporation of greater than 25 nucleotides. At 4 degrees C, switching can be calculated to occur in less than three nucleotide addition steps. Thus, with certain homopolymers, conditions can be found by which AMV DNA polymerase can switch from one template-primer complex to another, perhaps after each nucleotide addition step.

Avian Leukosis Virus↗
PubMed1976

On the fidelity of DNA replication. Enzyme activities associated with DNA polymerases from RNA tumor viruses.

DNA polymerase from RNA tumor viruses ("reverse transcriptase") has been analyzed for activities which have been associated with other DNA polymerases. Homogeneous DNA polymerase from avian myeoblastosis virus catalyzes pyrophosphate exchange and pyrophosphorolysis. Pyrophosphate exchange is dependent on a template and is base-specific. With avian myeloblastosis virus DNA polymerase, ribonucleotide templates are more efficient for synthesis while deoxyribonucleotide templates are more effective for pyrophosphate exchange. Synthesis, pyrophosphate exchange, and pyrophosphorolysis were inhibited by the chelating agent 1,10-phenanthroline, suggesting that enzyme-bound zinc is required for each of these reactions. The pyrophosphate exchange reaction was also demonstrated with the DNA polymerase from a mutant of Rous sarcoma virus that possesses a temperature-sensitive DNA polymerase. The pyrophosphate exchange reaction with the mutant polymerase is temperature-sensitive which demonstrates that pyrophosphate exchange is indeed catalyzed by the viral DNA polymerase and that the same mutation effects both DNA polymerase and pyrophosphatase activity. Unlike Escherichia coli DNA polymerase I, the DNA polymerase from avian myeloblastosis virus fails to degrade polydeoxyribonucleotides or to convert deoxynucleoside triphosphates into monophosphates. This lack of hydrolytic activities in avian myeoblastosis DNA polymerase should facilitate kinetic studies on the mechanism of DNA synthesis by this enzyme.

Avian Leukosis Virus↗
PubMed1976

On the fidelity of DNA replication. Lack of exodeoxyribonuclease activity and error-correcting function in avian myeloblastosis virus DNA polymerase.

Homogeneous DNA polymerase ("reverse transcriptase") from avian myeoblastosis virus was assayed for exodeoxyribonuclease activity. The substrates were defined template-initiator complexes in which different radioactive nucleotides were present at the 3'-OH termini of the initiator. Even when the number of molecules of enzyme was equal to the number of initiator termini there was no significant release of radioactivity with any of the template-initiator combinations tested. Under similar conditions, the nuclease activity associated with either Escherichia coli or T4DNA polymerases rendered more than 90% of the initiator termini acid-soluble. The ratio of exodeoxyribonuclease activity to protein with avian myeoblastosis DNA polymerase is less than 0.003% of that obtained with E. coli DNA polymerase I. Furthermore, avian myeloblastosis virus DNA polymerase failed to excise mispaired terminal nucleotides in both the presence and absence of polymerization.

Avian Leukosis Virus↗