RESUMEN
Four 3'-mercapto-2',3'-dideoxynucleoside 5'-triphosphates (A, G, C and T) were tested as DNA chain terminator substrates for calf thymus alpha-DNA polymerase, E. coli DNA polymerase I Klenow fragment, terminal deoxynucleotidyl transferase and reverse transcriptases of AMV, HIV and MLV viruses. It was shown that the analogues selectively and irreversibly terminated DNA chain elongation by AMV and HIV reverse transcriptases and the terminal transferase. Other DNA polymerases tested did not use the nucleotide analogues as chain terminator substrate.
Asunto(s)
ADN Viral/biosíntesis , VIH/efectos de los fármacos , Inhibidores de la Transcriptasa Inversa , Compuestos de Sulfhidrilo/farmacología , Secuencia de Bases , VIH/enzimología , Datos de Secuencia Molecular , Inhibidores de la Síntesis del Ácido NucleicoAsunto(s)
ADN Polimerasa Dirigida por ADN/metabolismo , Nucleótidos de Timina/metabolismo , Animales , Virus de la Mieloblastosis Aviar/metabolismo , Replicación del ADN , Indicadores y Reactivos , Estructura Molecular , ADN Polimerasa Dirigida por ARN/metabolismo , Relación Estructura-Actividad , Especificidad por Sustrato , Nucleótidos de Timina/síntesis químicaRESUMEN
The group of DNA polymerases was studied using some new nucleoside 5'-triphosphate analogs with termination substrate properties. Among DNA polymerases tested the least specific appeared reverse transcriptases of retroviruses and the most specific were DNA polymerases alpha type from high eucaryotes including mammalians.
Asunto(s)
ADN Polimerasa Dirigida por ADN/metabolismo , Desoxirribonucleótidos/síntesis química , Animales , Virus de la Mieloblastosis Aviar/enzimología , Bovinos , ADN/biosíntesis , Escherichia coli/enzimología , Hígado/enzimología , Ratas , Especificidad por Sustrato , Fagos T/enzimología , Timo/enzimologíaRESUMEN
A number of nucleoside 5'-triphosphate analogs were tested with Escherichia coli DNA polymerase I and Klenow fragment of the enzyme, bacteriophage T4 DNA polymerase and calf thymus DNA polymerase alpha. It was shown that 3'-amino-2',3'-dideoxynucleoside 5'-triphosphates as well as a number of 3'-derivatives of dTTP(3'NH2) are able to terminate DNA synthesis catalyzed by each enzyme if the reaction is performed in the absence of natural substrates. ddNTP and dNTP(3'F) were found to be inactive with DNA polymerase alpha only, but araNTP(3'NH2) was inactive with E. coli DNA polymerase I. dTTP(3'N3), dGTP(3'N'3), dCTP(3'N3), araNTP(3'N3) and (alpha-thio)dTTP(3'F) were unable to inhibit any of the above-mentioned DNA polymerases, in contrast to reverse transcriptase, accessible to the most nucleotide analogs tested.
Asunto(s)
ADN Polimerasa Dirigida por ADN/metabolismo , Nucleótidos/metabolismo , ADN/biosíntesis , ADN/metabolismo , Difosfatos/metabolismo , Hidrólisis , Inhibidores de la Síntesis del Ácido Nucleico , Nucleótidos/farmacología , Especificidad por SustratoRESUMEN
dNTP(3'-OCH3), a 3'-O-methyl derivative of dNTP, is a chain terminator substrate for DNA synthesis catalyzed by AMV reverse transcriptase. The enzyme seems to be the only DNA polymerase susceptible to the inhibitor while all the other DNA polymerases tested are fully resistant to the nucleotide analog. The resistant polymerases are: E. coli DNA polymerase I, Klenow's fragment of DNA polymerase I, phage T4 DNA polymerase, calf thymus DNA polymerase alpha, rat liver DNA polymerase beta and calf thymus terminal deoxyribonucleotidyl transferase.
Asunto(s)
Desoxirribonucleótidos/farmacología , Inhibidores de la Transcriptasa Inversa , Animales , Bovinos , ADN/biosíntesis , ADN Polimerasa I/antagonistas & inhibidores , ADN Polimerasa II/antagonistas & inhibidores , ADN Polimerasa Dirigida por ADN/metabolismo , Escherichia coli/enzimología , Hígado/enzimología , Inhibidores de la Síntesis del Ácido Nucleico , Oligonucleótidos/metabolismo , Ratas , Fagos T/enzimología , Timo/enzimologíaRESUMEN
It is shown that dNTP(3'F) are terminators of DNA synthesis and may serve as very effective tools for DNA sequencing with E.coli DNA polymerase I and AMV reverse transcriptase. The dNTP(3'F) are found to be chain terminator substrates for calf thymus terminal deoxyribonucleotidyl transferase but not for calf thymus DNA polymerase alpha. The optimal dNTP(3'F) concentration for DNA sequencing by DNA polymerase I is found to be an order of magnitude lower than that of ddNTPs. dNTP(3'F) produce a more clear sequence pattern than do ddNTPs.
Asunto(s)
Replicación del ADN/efectos de los fármacos , Desoxirribonucleótidos/farmacología , Flúor/farmacología , Animales , Secuencia de Bases , Bovinos , ADN Nucleotidilexotransferasa/metabolismo , ADN Polimerasa I/metabolismo , Escherichia coli , ADN Polimerasa Dirigida por ARN/metabolismoRESUMEN
It is shown that 2',3'-dideoxy-3'-aminonucleoside 5'-triphosphates with adenine, guanine, cytosine and thymine bases are effective inhibitors of DNA polymerase I, calf thymus DNA polymerase alpha and rat liver DNA polymerase beta. The effect of the above-mentioned compounds is markedly higher than corresponding action of the well-known DNA synthesis inhibitors arabinonucleoside 5'-triphosphates and 2',3'-dideoxynucleoside 5'-triphosphates. 2',3'-dideoxy-3'-aminonucleoside 5'-monophosphate residues incorporate into the 3'-terminus of the primer and terminate the DNA chain elongation. The possibility of using 2',3'-dideoxy-3'-aminonucleoside 5'-triphosphates as terminators for DNA sequencing by the polymerization method is demonstrated.