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Biomedical subjects

L A Loeb

Publications and source records attributed to L A Loeb.

At least 253 records · Page 14Linked to original sources
PubMed1976

Metal-induced infidelity during DNA synthesis.

The effect of several divalent cations on the accuracy of DNA replication in vitro has been examined. Only Be2+ altered the accuracy of DNA synthesis using purified DNA polymerase (DNA nucleotidyltransferase; deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase; EC 2.7.7.7) from avian myeloblastosis virus. The Be2+-induced base substitutions occurred with all templates and with all nucleotides tested. Analysis of the product by equilibrium density centrifugation and processive hydrolysis with snake venom phosphodiesterase suggested that the noncomplementary nucleotides were present in phosphodiester linkage. Nearest neighbor studies indicated that many of the Be2+-induced errors were present as single base substitutions. The enhancement of error frequency could be duplicated by the pretreatment of the enzyme, but not the template, with Be2+. Glycerol gradient centrifugation dissociated the Be2+-DNA polymerase complex and restored the initial error frequency of the polymerase. Thus, the weak binding of a metal cation to a DNA polymerase could alter the accuracy with which that polymerase copied DNA. Beryllium is a known carcinogen. The potential use of this system as a screening technique to detect chemical mutagens and carcinogens is considered.

Avian Myeloblastosis Virus↗
PubMed1976

Restriction of carcinogen-induced error incorporation during in vitro DNA synthesis.

The in vitro accuracy of DNA replication has been investigated through the measurement of the frequency with which noncomplementary nucleotides were incorporated during polynucleotide replication. The effect of beta-propiolactone treatment of deoxynucleotide templates, ribopolynucleotide templates, and the DNA polymerase from avian myeloblastosis virus was determined. Treatment of the deoxynucleotide template, poly(dA) (see article) oligo(dT) 12-18, by beta-propiolactone resulted in an increased frequency of noncomplementary nucleotide incorporation during DNA polymerization. Carcinogen treatment of the ribonucleotide templates, poly(rA) (see article) oligo(dT) 12-18, and poly(rC) (see article) oligo(dG) 12-18, and carcinogen treatment of avian myeloblastosis virus DNA polymerase did not alter the frequency of noncomplementary nucleotide incorporation. This suggested that carcinogen-induced error incorporation during DNA synthesis was restricted solely to the treatment of a deoxynucleotide template.

Avian Myeloblastosis Virus↗
PubMed1975

Conformation of deoxynucleoside triphosphate substrates on DNA polymerase I from Escherichia coli as determined by nuclear magnetic relaxation.

A unique conformation of deoxynucleoside triphosphate substrates bound to Escherichia coli DNA polymerase I has been determined by nuclear magnetic resonance techniques. The effects of Mn(II) bound at the active site of the enzyme on the longitudinal (T1p-1) and transverse (T2p-1) relaxation rates of the alpha, beta, and gamma phosphorus atoms and 5 protons of enzyme-bound thymidine 5'-triphosphate (dTTP) were measured at 40.5 MHz (31P), 100 and 220 MHz (1H). From frequency dependence of T1p-1, a correlation time of 7 X 10(-10) s and Mn(II) to proton distances of 10.4, 9.9, 10.3, 10.8, and 8.4 A were calculated for the --CH3, H6, H'1, H'2, and H'4 protons. The calculated Mn(II) to phosphorus distances of 4.2, 4.8, and 3.2 A for the alpha, beta, and gamma phosphorus atoms indicates that Mn(II) corrdinates directly only with the gamma-phosphoryl group and that a puckered triphpsphate conformation exists for the enzyme-bound dTTP. This differs from the binary Mn(II)-dTTP complex in which alpha, beta, and gamma phosphoryl coordination occurs, and a thymine-deoxyribose torsion angly (chi) about the glycosidic bond of 40 degrees is detected. The eight manganese-substrate distances on the enzyme are fit by a unique Mn-dTTP conformation, with a torsion angle equal to 90 degrees, indistinguishable from that found for a deoxynucleotidyl unit in double helical DNA-B. Hence, binding to DNA polymerase appears to adjust the conformation of dTTP for Watson-Crick basepairing. Similarly, the binding of Mn-dATP to DNA polymerase I increased the distances from Mn(II) to the H2, H8, H'1, and H'4 protons of dATP but the adenine-deoxyribose torsion angle of 90 degrees was preserved. Such preorientation of substrates could facilitate incorporation of the complementary nucleotide. When positioned within the DNA structure, the conformation of enzyme-bound Mn-dTTP requires an inline nucleophilic attack on the alpha phosphorus with Mn(II) promoting pyrophosphate departure.

Binding Sites↗
PubMed1975

Kinetic analysis of Escherichia coli deoxyribonucleic acid polymerase I.

The kinetic properties of Escherichia coli DNA polymerase I were simplified to those of a 1 deoxynucleotide substrate reaction by the use of polynucleotide templates. With poly(dA)-oligo(dT) as the template-primer complex, Mg2+ decreases the Km of the substrate dTTP but has little or no effect on the Km of the substrate Mg-dTTP, suggesting that multiple pathways involving the binding of Mg2+, dTTP, and Mg-dTTP are operative in forming the active complex. The Km of free Mg2+, extrapolated to zero concentration of substrate (830 = 62 muM), agrees within a factor of 2 with the dissociation constant of magnesium from 4 +/- 1 sites on the enzyme determined previously by binding studies (Slater, J.P., Tamir, I., Loeb, L.A., and Mildvan, A.S. (1972) J. Biol. Chem. 247, 6784-6794). The maximal turnover number with poly(dA) as template is 5.7 +/- 0.7 s-1. Changing the nature of the base in the polydeoxynucleotide template alters the maximal rate of polydeoxynucleotide synthesis by an overall factor of 31 with poly(dC) is greater than poly(dT) is greater than poly(dA) is greater than poly(dG), indicating that pyrimidine templates are copied faster than purine templates. Changing the sugar structure from poly(dA) to poly(rA) causes a 3-fold increase in the rate of template copying. A study of the kinetic effects of all noncomplementary deoxynucleotides with all deoxynucleotide templates, as well as with poly(rA)-oligo(dT), yields complex patterns of activation and inhibition requiring from 1 to 2 additional binding sites for the noncomplementary nucleotides. The kinetically determined affinities of the active site of the enzyme-template-primer complex for the complementary free nucleotide (as measured by Km) generally exceed those for the noncomplementary neuclotides (as measured by KI slope) by 1 or more than 3 orders of magnitude.

Binding Sites↗
PubMed1975

Avian myeloblastosis virus DNA polymerase. Kinetic studies on the incorporation of noncomplementary nucleotides.

The high error rate characteristic of DNA polymerases from RNA tumor viruses has permitted measurements on the simultaneous incorporation of complementary and noncomplementary nucleotides during DNA synthesis. For example, avian myeloblastosis virus DNA polymerase incorporates 1 molecule of dCMP for approximately 500 molecules of dTMP polymerized using polyriboadenylic acid as a template. The parallel incorporation of complementary and noncomplementary nucleotides afer gel filtration of avian myeloblastosis virus DNA polymerase indicates that the observed fidelity is catalyzed by the polymerase itself. Nearest neighbor analysis of the product indicates that noncomplementary nucleotides are incorporated as single base substitutions. The incorporation of the noncomplementary dCMP is not reduced by a 20-fold greater amount of the complementary nucleotide, dTTP. Conversely, the concentration of the noncomplementary nucleotides does not effect the rate of incorporation of the complementary nucleotide. A similar lack of competition between complementary dGTP and noncomplementary dATP is exhibited using poly(rC)-oligo(dG) as a template-primer. Furthermore, there was no detectable competition between the different noncomplementary nucleotides. Possible explanations for this lack of competition are considered.

Avian Leukosis Virus↗
PubMed1975

On the fidelity of DNA replication. Characterization of polynucleotides with errors in base-pairing synthesized by avian myeloblastosis virus deoxyribonucleic acid polymerase.

Polynucleotide templates were copied by avian myeloblastosis virus DNA polymerase ("reverse transcriptase") and the frequency and distribution of errors were determined. The error rate with [r(pA)2500-d(pT)12-18] template-initiator under a variety of conditions was approximately 1/600, i.e. one incorrect dCMP incorporated for 600 correct dTMP polymerized. Addition of the metal chelator o-phenanthroline to the reaction inhibited the incorporation of correct and incorrect nucleotides proportionately. The enzyme inhibited a pH optimum of 8.5 and the error rate remained constant over a range of pH (6.0 to 10.0). The rate of polymerization was greater at higher temperatures and approximately doubled for every 10 degrees increase. The error rate was constant at all temperatures. These results indicate that the purified avian myeloblastosis virus DNA polymerase synthesizes polydeoxynucleotides with an unusually large number of errors in base-pairing. Velocity sedimentation of the products of the reaction obtained at various times during the course of synthesis indicate that: (a) the entire length of the 14 S template was copied, and (b) the incorporation of the incorrect nucleotide did not terminate chain propagation. Isopyknic banding in neutral and alkaline cesium sulfate gradients showed that the noncomplementary nucleotides are incorporated into the polydeoxynucleotide product. Stepwise degradation and nearest neighbor analysis of the reaction product indicated that (a) the correct and incorrect nucleotides are present in phosphodiester linkages, (b) the errors are not concentrated at either termini; and (c) the errors are uniformly distributed throughout the newly synthesized polydeoxynucleotide.

Avian Leukosis Virus↗
PubMed1974

Reverse transcriptase: correlation of zinc content with activity.

Evidence is presented that DNA polymerase of avian myeloblastosis virus has an obligatory zinc requirement for activity. Previous studies indicate that the purified polymerase contains zinc in a stoichiometry of about 1 g-atom/mole. We now find that the enzyme-bound zinc is exchangeable with radioactive (65)Zn; after isoelectric focusing, the radioactive (65)Zn is coincident with polymerase activity. Dialysis of the (65)Zn-labeled polymerase against the chelator, 1,10-phenanthroline, results in a progressive loss of radioactive (65)Zn and polymerase activity. Thereupon, incubation of the inactivated enzyme with Zn(2+) fully restores activity. Thus, the DNA polymerase present in an oncogenic RNA virus, like animal DNA polymerases, can be rigorously classified as a zinc metalloenzyme. DNA polymerase of avian myeloblastosis virus is inactivated by 1,10-phenanthroline at a much faster rate than the bacterial and animal DNA polymerases that have been tested. It may, therefore, be possible to inactivate selectively DNA polymerases from animal tumor viruses by brief exposure to appropriate metal chelators.

Avian Leukosis Virus↗