pyrophosphate and 2--3--dideoxythymidine-triphosphate

A simple and sensitive technique for detection of reverse transcriptase (RT) activity in real time has been developed. The technique is based on continuous detection of the inorganic pyrophosphate formed in the RT-catalyzed reaction by a luminometric method. The technique has been used for continuous monitoring of RT-catalyzed DNA synthesis on both homo- and heteropolymeric templates. The assay is sensitive and yields linear responses between 1.5-960 mU of avian myeloblastosis virus RT (AMV-RT). The assay was used for detection of the inhibitory effect of dideoxythymidine (ddTTP) on the AMV-RT activity and also for real-time detection of single-base incorporation events catalyzed by AMV-RT. The possibility of using the new technique for other applications is discussed.

Topics: Avian Myeloblastosis Virus; Deoxyribonucleotides; Dideoxynucleotides; Diphosphates; DNA; Kinetics; Luminescent Measurements; Reverse Transcriptase Inhibitors; RNA-Directed DNA Polymerase; Thymine Nucleotides

In connection with the characterization of two DNA polymerases (DPols) of Chlorella, we have extensively surveyed the literature on inhibition studies on DPols in various eukaryotes. By applying Tamiya's plot (1), we have obtained two parameters for each of the inhibitors, phi- and n-values, which express the enzyme sensitivity to the drug and the number of inhibitor molecules present in the enzyme-inhibitor complex that is principally involved in the inhibition, respectively. By inspecting these parameters for the three mammalian DPols, alpha-, beta-, and gamma-pols, as well as other eukaryotic DPols, we have found that: [1] inhibitors commonly utilized for characterizing various DPols can be classified into two major groups, each having two subgroups, on the basis of a comparison of the phi values among alpha-, beta-, and gamma-pols. Moreover, the grouping seems not to be merely coincidental, but to be intrinsically related to facets of the enzyme reaction, which may be taken to reflect evolutionary differences in DPol structure and function among the three DPols; [2] the remarkable n value, n = 1/2, that has been found for the inhibitors competitive with dCTP in Chlorella DPols has also been detected widely in many other eukaryotic DPols. Based on the first finding as well as many other data on various DPols, we have proposed an evolutionary scenario for eukaryotic DPols. Based on the second finding, we have hypothesized a novel role for dCTP as a cofactor, probably an apparent allosteric effector, in the nucleotide transfer reaction mechanism.

Topics: Aphidicolin; Arabinofuranosylcytosine Triphosphate; Biological Evolution; Cations, Divalent; Chlorophyta; Deoxycytosine Nucleotides; Dideoxynucleotides; Diphosphates; Diterpenes; Ethidium; Ethylmaleimide; Nucleic Acid Synthesis Inhibitors; Thymine Nucleotides