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Biomedical subjects

L A Loeb

Publications and source records attributed to L A Loeb.

At least 199 records · Page 11Linked to original sources
PubMed1983

Mammalian mutator mutant with an aphidicolin-resistant DNA polymerase alpha.

The Chinese hamster V79 cell mutant aphr-4-2, selected for its resistance to aphidicolin, a specific inhibitor of DNA polymerase alpha (DNA nucleotidyltransferase, EC 2.7.7.7), is characterized by slow growth, UV sensitivity, and hypersensitivity to UV-induced mutation. DNA polymerase alpha has been purified from mitochondria-free crude extracts of the mutant and its parental wild-type cells by sequential column chromatography on DEAE-cellulose and phosphocellulose. The major DNA polymerase activity from both cell lines was found to have characteristics of the alpha-type polymerase: sensitivity to 0.2 M KCl, resistance to heat denaturation (45 degrees C for 15 min), an apparent Km of 5 microM for dATP, and an ability to copy poly(dT)X(rA)10 but not poly(rA)X(dT)12. The crude extracts and purified DNA polymerase alpha from the mutant cells are not inhibited by aphidicolin (greater than 0.6 microM). The apparent Km for dCTP with DNA polymerase alpha is 1.0 +/- 0.4 microM (mean +/- SD) for the mutant enzyme. The polymerase from the parental cells, similarly purified, is sensitive to aphidicolin and has an apparent Km for dCTP of 10 +/- 4 microM. The spontaneous mutation rate (per cell per division), determined by fluctuation analysis at the Na+/K+-ATPase (EC 3.6.1.8) locus, is higher for mutant cells (42-73 x 10(-8)) than for parental cells (3-16 x 10(-8)). These data suggest a mechanism for aphidicolin resistance of the mutant--i.e., a decrease in the Km for dCTP. The results also indicate that an altered DNA polymerase alpha may be intrinsically mutagenic during normal semiconservative replicative as well as during UV-induced repair syntheses.

Animals↗
PubMed1983

Mechanisms of neoplastic transformation.

This paper introduces a series of invited essays on current controversies in basic cancer research. The initial group of essays focuses on the detection and interpretation of molecular and cellular changes suspected to be of importance in the cause and pathogenesis of cancer. There are two formats: (i) differing viewpoints are presented in parallel, or (ii) the author(s) evaluate a hypothesis in light of available data. Each type of paper aims to critically evaluate current hypotheses and supporting data, while avoiding pronouncements on validity. Recent advances in molecular biology now permit us to consider genes as chemical entities. Individual genes can be isolated, cloned to produce multiple copies, sequenced, and assayed for biological function. This new molecular technology is being applied to fundamental questions in cancer research. The controversies resulting from these pioneering studies are the topics of the initial papers in this series. Forthcoming essays will concern the mechanism(s) of tumor promotion; a search for cancer genes by DNA transfection; the role of DNA rearrangements as initiating events in carcinogenesis; the O6 position of guanine as a critical target of carcinogens; and metals as mutagens and carcinogens.

Animals↗
PubMed1983

Infidelity of DNA synthesis as a cause of mutagenesis.

The concept underlying these studies is that a major determinant of mutagenesis involves perturbations in the fidelity of DNA replication. i.e., the accuracy by which DNA polymerases copy DNA templates. To investigate this relationship, we have designed in vitro assays to measure the accuracy of DNA replication and used these systems to screen for and to quantitate factors that promote errors in DNA synthesis. Using DNA polymerase from bacteria, the frequency of mistakes with phi X174 DNA as a template approaches 10(-7) and is similar to the spontaneous mutation rates in bacterial cells. In contrast, DNA polymerases from animal cells are more error-prone. The differences in fidelity among mammalian DNA polymerases which lack error-correcting mechanisms suggest that these enzymes enhance accuracy by improving base-selection. Thus, mutants in DNA polymerase-alpha might be altered in base-selection. Chinese hamster V79 cell mutants selected by resistance to aphidicolin, a specific inhibitor of DNA polymerase-alpha, have been reported (Somatic Cell Genet., 7: 235-253, 1981). DNA polymerase-alpha was purified from mitochondria-free crude extracts of these mutants by sequential column chromatography using DEAE-cellulose and phosphocellulose. DNA polymerase-alpha purified from one of the mutants is 10-fold more resistant to aphidicolin than the same enzyme purified from the parental cells. Moreover, the apparent Km for dCTP is 1.0 +/- 0.4 microM for the mutant polymerase and 10 +/- 4 microM for the parental enzyme. These observed differences are in accord with the known competition between aphidicolin and dCTP, and provide a mechanism for the aphidicolin resistance of the mutant, i.e., the decrease in Km for dCTP. The elevated spontaneous and induced mutation rate exhibited by this mutant could be mediated by the alteration in DNA polymerase-alpha. With DNA replicating enzymes from a variety of sources, enhancement of mutagenesis has been demonstrated by alteration in precursor pools, damage to DNA templates, loss of nucleotide bases on DNA, metal ions that interact with nucleotide bases, and organic compounds that intercalate into DNA. The alterations of deoxynucleoside triphosphate pools also occur after treatment of animal cells with known mutagens. This observation may provide a new mechanism for mutagenesis by these agents independent of alterations in DNA.

Animals↗
PubMed1983

Effect of hyperthermia on the survival of normal human peripheral blood mononuclear cells.

Human peripheral blood mononuclear cells from normal healthy volunteers were exposed to elevated temperatures of 41-43 degrees for up to 6 hr. Thereafter, the cells were stimulated with phytohemagglutinin in vitro in order to measure indirectly the surviving fraction. DNA replication in heated cells in response to phytohemagglutinin was found to be a sensitive indicator of thermal injury. Exposure to even 40 degrees for 2 hr lowered thymidine incorporation at early time points after phytohemagglutinin stimulation, but the cells were able to recover from thermal injury after exposure for up to 4 hr at 42 degrees. At 43 degrees, exposure for even 1 to 2 hr caused irreversible damage. The changes in thymidine incorporation were not due to changes in endogenous nucleotide pools since parallel changes were observed in DNA polymerase activity. Thus, the heat sensitivity of normal human lymphocytes could be a limiting factor for use of hyperthermia as an adjunct to radiotherapy and chemotherapy of human cancer.

Cells, Cultured↗
PubMed1982

Depurination of DNA as a possible mutagenic pathway for cells.

The possible consequences of depurination for both spontaneous and induced mutagenesis were investigated using in vitro and in vivo assays. Depurination of synthetic polynucleotide templates such as poly [d(A-T)] or poly [d(G-C)] leads to increased misincorporation of noncomplementary nucleotides when these templates are copied by prokaryotic and eukaryotic DNA polymerases. The ability of Escherichia coli DNA polymerase I to copy over apurinic sites was demonstrated using single-stranded circular DNA of bacteriophage 0X174 as a template and starting DNA synthesis at a fixed point. Analysis of the newly synthesized 0X174 restriction fragments on neutral and alkaline sucrose gradients shows that synthesis proceeded past apurinic sites. When using depurinated 0X174 DNA containing the am3 amber mutation as a template for copying by E. coli DNA polymerase I, an increased reversion to wild type is observed after transfection into E. coli spheroplasts. The enhancement in reversion frequency is proportional to the extent of depurination, suggesting that depurination is also mutagenic during copying natural DNA in vitro. When noncopied depurinated 0X174 am3 DNA is transfected in E. coli spheroplasts, no increase in reversion frequency is observed above background level. However, when the spheroplasts are derived from bacteria in which the SOS response had been induced by UV irradiation, a substantial increase is observed for depurinated molecules, whereas no increase is observed for nondepurinated templates, suggesting in vivo mutagenesis at depurinated sites. In each of the different assay systems investigated, the increase in misincorporation or reversion frequency is a linear function of the number of sites and is abolished by treatment of the depurinated templates with alkali, which rapidly induces strand breakage at apurinic sites.

Apurinic Acid↗
PubMed1982

Mutagenesis resulting from depurination is an SOS process.

When bacteriophage phi X174 am3 DNA depurinated in vitro is transfected into E. coli spheroplasts prepared from bacteria previously exposed to UV light, a strong mutagenic response is observed. This mutagenic response does not occur in spheroplasts derived from pre-irradiated bacteria carrying defective recA, recF or umuC genes. These findings indicate that mutagenesis at apurinic sites is an SOS-dependent process. The mutagenic response is not dependent on the multiplicity of transfection. This suggests that mutagenesis is not mediated by recombination.

Bacteriophage phi X 174↗
PubMed1982

The mutagenic effect of deoxynucleotide substrate imbalances during DNA synthesis with mammalian DNA polymerases.

The frequency of reversion of phi X174 amber mutants to wild-type, resulting from in vitro DNA synthesis catalyzed by eucaryotic DNA polymerase-alpha or -beta, varies over a 10- to 1000-fold range. This variation is dependent on the relative ratio of deoxyribonucleotide substrates present during in vitro DNA synthesis. The effect is observed at two different loci in the genome and with several different DNA polymerases. In addition, the effect is observed using an unfractionated cellular extract. These results provide support for the hypothesis that altered nucleotide pools cause mutations in mammalian cells by decreasing the fidelity of DNA synthesis.

Bacteriophage phi X 174↗
PubMed1982

Role of depurination in mutagenesis by chemical carcinogens.

The effect of modifying phi chi 174 viral DNA by the chemical carcinogens beta-propiolactone, N-acetoxyacetylaminofluorene and anti-benzo[a]pyrene diol-epoxide was investigated by transfecting the modified DNA into Escherichia coli spheroplasts. Modification of the DNA in vitro by each of these agents was mutagenic for the phi chi 174 amber mutants am3 and am86. Mutagenicity depended on the induction of the "SOS" response in the host spheroplasts. Heating beta-propiolactone-treated DNA at neutral pH caused strong inactivation such that the number of lethal hits was increased 4-fold. Sucrose gradient analysis showed the induction of alkali-labile sites in the heated DNA. The "nicked circle assay" with double-stranded phi chi 174 DNA showed greater than 70% of these sites to be apurinic sites. Concomitantly with the production of these new sites, a strong increase in the mutation frequency was observed. This mutagenesis also depended upon the induction of the error-prone SOS response in the spheroplasts, as was previously shown to be the case for mutagenesis at putative apurinic sites induced directly by acid-heat treatment. These results suggest that depurination may be of importance to the mechanism of mutagenesis by beta-propiolactone and other carcinogens.

Acetoxyacetylaminofluorene↗
PubMed1981

S1 nuclease does not cleave DNA at single-base mis-matches.

Three assays have been designed to detect the cleavage of duplex phi X174 DNA at single-base mis-matches. Studies with S1 nuclease failed to detect cleavage at mis-matches. S1 nuclease digestion at 37 and 55 degrees C failed to produce a preferential degradation of a multiply mis-matched heteroduplex when compared to a mis-match-free homo-duplex as analyzed by sedimentation on sucrose gradients. Other heteroduplex templates were not cleaved by S1 nuclease at a defined single-base mis-match when assayed by gel electrophoresis or by marker rescue. In all cases, the amount of S1 nuclease employed was at least 10-times more than that required to render a single-stranded phi X174 DNA molecule completely acid soluble. The rate of hydrolysis of single-base mis-matches by S1 nuclease was estimated to be less than 0.016% of the rate at a base in single-strand phi X174 DNA. In no instance did we detect activity by S1 nuclease directed at mis-matched sites in our template molecules. Similarly, the single-strand specific endonuclease from Neurospora crassa does not cleave heteroduplex templates at a defined single-base mis-match when assayed by marker rescue.

Bacteriophage phi X 174↗
PubMed1981

On the fidelity of DNA replication. Effect of the next nucleotide on proofreading.

The contribution of proofreading to the fidelity by which Escherichia coli DNA polymerase I copies natural DNA has been analyzed by two independent criteria. With phi X174 am 3 DNA as a template, there is approximately a 25-fold increase in noncomplementary base substitutions at position 587 when the concentration of the next correct nucleotide, dATP, is increased. Sequence analysis indicates that the mistakes represent misincorporation of C in place of T at position 587. This mutagenic response is presumed to result from a decrease in the probability of excision by the 3' leads to 5' exonuclease of Pol I and is considered within the context of current theories on proofreading. No enhanced mutagenicity is observed with avian myeloblastosis virus DNA polymerase, which lacks a 3' leads to 5' exonuclease. Using a second approach, an enhancement in mutagenesis as large as 30-fold is observed to result from the addition of deoxynucleoside monophosphates to the Pol I reaction. This mutagenicity occurs with any of the four deoxynucleoside monophosphates and is independent of a significant inhibition of DNA synthesis, thus supporting proofreading models in which sites of excision and incorporation are independent. The results of both approaches suggest that the exonucleolytic activity of Pol I can increase fidelity by approximately 30-fold on natural DNA, a value much higher than previous estimates with polynucleotide templates. The effect of the next correct nucleotide in decreasing accuracy provides an in vitro probe for screening eukaryotic cells for putative proofreading functions.

Bacteriophage phi X 174↗
PubMed1981

Fidelity of mammalian DNA polymerases.

The fidelity of copying natural DNA in vitro with each of the three classes of eukaryotic DNA polymerases has been determined. DNA polymerases-beta and -gamma are highly inaccurate, catalyzing noncomplementary single-base substitution at a frequency between 1/3000 and 1/8000. DNA polymerase-alpha is substantially more accurate, with an error rate of 1/30,000. When the error rates of these DNA polymerases are considered in the context of the accuracy of DNA replicative processes in vivo, it seems likely that other factors must exist in mammalian cells which are involved in the accurate replication and maintenance of the genetic information.

Animals↗
PubMed1981

On the fidelity of DNA replication. Nucleoside monophosphate generation during polymerization.

During catalysis by homogeneous procaryotic DNA polymerases, nucleoside monophosphates are generated by a 3' leads to 5'-exonucleolytic activity. Using Escherichia coli DNA polymerase I and poly[d(A-T)] as a template, the contribution of this activity to the fidelity of DNA synthesis has been evaluated by three different criteria. 1) The ratio between the rates of monophosphate generation and incorporation of the noncomplementary nucleotide with Mg2+ as an activating cation was 0.6 +/- 0.6, which is insufficient to account for the high fidelity of polymerization. 2) Inhibition of polymerization by pyrophosphate fails to diminish fidelity, although some kinetic models suggest that optimal error correction via monophosphate release requires the polymerization reaction to be strongly driven by pyrophosphate release. 3) The addition of deoxynucleoside monophosphates in concentrations as great as 10 mM to the reaction mixture does not alter the fidelity of DNA synthesis. These observations argue against the kinetic proofreading mode to account for the fidelity of E. coli DNA polymerase I when copying poly[d(A-T)] in a Mg2+-activated reaction. Furthermore, they suggest that the polymerase may enhance specificity at the base-selection step. However, the 3' leads to 5' exonuclease plays a larger role when the polymerase is activated with Mn2+ and may also be important in copying natural DNA where lower error rates are observed in vitro.

DNA Polymerase I↗
PubMed1981

On the activity and fidelity of chromatin-associated hepatic DNA polymerase-beta in aging murine species of different life spans.

Activity and accuracy of chromatin-directed DNA replication have been compared in young and aged Mus musculus and Peromyscus leucopus, two murine species with contrasting maximum lifespans. Chromatin isolated from livers of mature adults of both species copied efficiently exogenous DNA templates using predominantly DNA polymerase-beta. The DNA synthetic activity of liver chromatin remained constant in both species throughout their lifetimes. The fidelity of chromatin-directed poly [d(A-T)] synthesis was similar for the comparatively short-lived M. musculus and the relatively long-lived P. leucopus and remained unaltered in old animals. The fidelity of poly [d(A-T)] copying catalyzed by DNA polymerase-beta-dissociated from liver chromatin was comparable to that of the chromatin-directed synthesis. The dissociation enzymes did not exhibit diminished fidelity of poly [d(A-T)] synthesis with age. In all ages of both species examined, the murine liver DNA polymerase-beta, both chromatin-associated and solubilized, exhibited high error frequencies; approximately one dGMP was incorporated for every 500-1,000 complementary nucleotides polymerized. The relationship of these results to the accuracy of DNA replication and repair as a determinant of aging is considered.

Aging↗