RESUMEN
We have investigated the ability of some nucleoside 5'-triphosphate analogues to terminate the DNA synthesis catalyzed by calf thymus DNA polymerase alpha and terminal deoxynucleotidyl transferase, rat liver DNA polymerase beta, E. coli DNA polymerase I (Klenow's fragment) and AMV reverse transcriptase. It has been shown that lyxoanhydronucleoside 5'-triphosphates terminate DNA synthesis catalyzed by reverse transcriptase and terminal deoxynucleotydil transferase. 2',3'-O-Isopropylidenecytidine 5'-triphosphate inhibits the DNA synthesis catalyzed by reverse transcriptase and DNA polymerase beta and its moiety was incorporated in the place of dTMP residue. Riboanhydroadenosine 5'-triphosphate reveals the properties of an effective termination substrate for all the DNA polymerases studied. This is the first attempt to investigate nucleotide analogues with the restricted conformation of the carbohydrate moiety as termination substrates for several prokaryotic and eukaryotic DNA polymerases.
Asunto(s)
ADN Polimerasa I/metabolismo , ADN/biosíntesis , Conformación de Ácido Nucleico , Nucleótidos/metabolismo , Animales , Secuencia de Bases , ADN Nucleotidilexotransferasa/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , Datos de Secuencia Molecular , ADN Polimerasa Dirigida por ARN/metabolismo , Especificidad por SustratoRESUMEN
The reaction of pyrophosphorolysis catalyzed by Escherichia coli DNA polymerase I Klenov fragment, calf thymus DNA polymerase alpha, rat liver DNA polymerase beta and AMV reverse transcriptase was studied. Some pyrophosphate (PPi) analogs were taken as low molecular weight substrates. It was shown that only imidodiphosphonic acid acted as the PPi substrate analog for the reactions catalyzed by DNA polymerases I and alpha, both imidodiphosphonic acid and methylenediphosphonic acid were active in the case of DNA polymerase beta and reverse transcriptase. Other analogs tested were neither nucleotide residue acceptors, nor inhibitors of the pyrophosphorolysis reaction with PPi. The abilities of some PPi analogs to inhibit the DNA elongation catalyzed by reverse transcriptase were investigated. The principles of specificity of low molecular substrates recognition by DNA polymerases and some problems concerning the mechanisms of DNA synthesis inhibition by PPi analogues are discussed.
Asunto(s)
ADN Polimerasa Dirigida por ADN/metabolismo , Difosfatos/metabolismo , Animales , Catálisis , Bovinos , Cromatografía en Capa Delgada , ADN/metabolismo , Escherichia coli/enzimología , Hidrólisis , Hígado/enzimología , Ratas , Especificidad por Sustrato , Timo/enzimologíaRESUMEN
Reaction of DNA synthesis catalyzed by DNA polymerase I KF in the presence of 2'-deoxynucleoside 5'-alpha-thiotriphosphates (dNTP alpha S) was investigated. DNA with thiophosphate groups (DNA[P=S]) obtained by such a way was studied in reactions of hydrolysis and pyrophosphorolysis catalyzed by DNA polymerase I KF. It is shown that the rate of DNA elongation is decreased both on the step of incorporation of dNMP alpha S residues and on the step of incorporation of the next dNMP residue. The rate of pyrophosphorolysis of 3'-terminal dNMP alpha S was demonstrated to be one order of magnitude less in comparison with the corresponding reaction with the natural dNMP residue. Contrary, the rate of 3'----5'-exonuclease hydrolysis of both DNA[P=S] and DNA of the same structure revealed no distinguishable differences.
Asunto(s)
ADN Polimerasa I/metabolismo , ADN Viral/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , Escherichia coli/enzimología , Bacteriófagos/genética , Secuencia de Bases , Cromatografía , Electroforesis en Gel de Poliacrilamida , Hidrólisis , Datos de Secuencia Molecular , Compuestos Organotiofosforados/metabolismo , FosforilaciónRESUMEN
2',3'-Dideoxy-2',3'-dehydrothymidine 5'-triphosphate (dddTTP) reveals the termination substrate properties in the DNA synthesis catalyzed by E. coli polymerase I (Klenow fragment), rat liver DNA polymerase beta, calf thymus terminal deoxynucleotidyl transferase, and reverse transcriptase of avian myeloblastosis virus but does not affect calf thymus DNA polymerase alpha. For DNA polymerase I, dddTTP by an order of magnitude is more effective than any known termination substrate. It is supposed that dddTTP models the conformational state of the substrate's carbohydrate moiety in the complex DNA polymerase + template-primer.