Evidence for Allosterism in In Vitro DNA Synthesis on RNA Templates

Abstract

Hemoglobin mRNA and (rA)n. (dT)lo have been used as primer-templates in a kinetic study of DNA synthesis with Escherichia coli DNA polymerase I (DNA nucleotidyl transferase, EC 2.7.7.7) and Mason-Pfizer monkey virus reverse transcriptase (RNA-directed DNA polymerase). The rate versus enzyme concentration curve is sigmoidal and is consistent with a cooperative phenomenon. The results could be interpreted in terms of the formation of an active complex containing enzyme dimers (or oligomers) on the primer-template. We have also observed sigmoidal kinetics in rate versus deoxynucleotide triphosphate concentration. These results are consistent with an allosteric mechanism in which the triphosphates act as both modifiers and DNA precursors. In the critical range, a 6-to 8-fold increase in both enzyme and triphosphate concentrations can lead to a 1500-fold increase in the rate of synthesis on an RNA template. Thus, small changes in enzyme and precursor concentrations could play a regulatory role in vivo.The complexity of the process of polymerization by various DNA polymerases (DNA nucleotidyl transferases, EC 2.7.-7.7) is only partially understood. There are at least five components to the system, namely, primer, template, enzyme, deoxynucleoside triphosphates, and metal ions. Studies of physical interactions between the enzyme and other individual components of the reaction, such as the work of Loeb and coworkers (1) on Escherichia coli DNA polymerase I-Mn++ complexes, and of Kornberg and coworkers (2-4) on the binding sites of E. coli DNA polymerase I, have contributed to an understanding of the system. Such studies are necessarily limited in their bearing on the synthetic process as a whole. In order to learn more about the nature of the interactions which take place during DNA synthesis, all components of the reaction must be present. We have therefore undertaken a kinetic study of DNA synthesis using DNA and RNA templates with E. coli DNA polymerase I. We report in this paper the effect of both polymerase and deoxynucleoside triphosphate concentrations on the rate of DNA synthesis. Rather than simple saturation kinetics we have observed sigmoid curves with RNA primer-templates (as opposed to DNA templates), suggesting a cooperative mechanism. MATERIALS AND METHODSUnlabeled deoxynucleoside-5'-triphosphates, (rA)0-(dT)lo, (dA)n (dT)lo, and (dC)0. (dG)12 were purchased from P-L Biochemical Laboratories; labeled triphosphates (18 Ci/mmol) were purchased from Schwarz-Mann. The homopolymer pairs were reannealed by heating at 55-60' for (rA)n0 (dT)lo and (dA).* (dT)lo and at 800 for (dCn*v (dG)12 for 15 min, in 0.05 M Tris, pH 7.8. The mixtures were then allowed to cool slowly to room temperature. The preparation of calf-thymus DNA has been described (5). Activated DNA was prepared according to the procedure of Aposhian and Kornberg (6). Hemo

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“…The sigmoidal nature of the plots shown in Figure S5 b,cindicates that the VE CeO 2 NPs act as multivalent or cooperative catalysts. [12] At certain concentrations,t he interactions between the nanoparticles appear to play an important role in enhancing the rate of the reaction. Such catalyst-dependent sigmoidal behavior has been ascribed to the formation of enzyme dimer or oligomers.…”

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“…the active site may be altered by interaction of the effector tRNA at a site on the enzyme which is not accessible in the oligomer. We have presented evidence for allosteric effects in the E. coli Pol I system (20).…”

Apparent allosterism by avian myeloblastosis virus reverse transcriptase and E coli DNA polymerase I

“…The kinetics of RNA directed DNA synthesis catalyzed by Mason-Pfizer monkey virus reverse transcriptase and by E. coli DNA polymerase I were recently studied by Cavalieri et al (1) using both homopolymers and heteropolymers as templates. A homopolymer, usually oligo(dT)10, was used as a primer.…”

“…The rate of synthesis also was Nucleic Acids Research found to increase sigmoidally as the concentration of enzyme was increased. Their data, obtained by using trichloroacetic acid (TCA) precipitation to isolate the product DNA, were interpreted as evidence for allosterism during reverse transcription (1). If Enzymes.…”

“…Nor are polymerase molecules under these conditions involved in cooperative interaction during reverse transcription. We believe that we have investigated the apparent cooperative effect previously described (1), and that the suggestion of allosterism was made on the basis of artifactual data. We suggest that in synthesis primed by oligo(dT)12 18 the polymerase does not act processively, and that the consequent end addition of a small number of nucleotides per available primer results in poor acid precipitation of the product, thus giving the appearance of cooperative behavior.…”