[Unprimed synthesis of poly (d(A-T)), catalyzed by a preparation of Escherichia coli DNA polymerase I].

Abstract:

The initial events of the de novo synthesis of poly[d(A-T)], catalyzed by preparations of E. coli DNA-polymerase I, were investigated. The data provide evidence that deoxynucleoside diphosphate: oligonucleotide deoxynucleotidyl transferase (dNDP-transferase), the enzyme which is able to catalyze unprimed polymerization of dNDP, participates in the process of initiation. This conclusion is based on the following data: 1) preincubation of E. coli DNA-polymerase I preparation with dADP and dTDT abolishes a lag-period in the poly[d(A-T)] synthesis; 2) dithiothreitol and N-ethylmaleinide, inhibitors of dNDP-transferase, inhibit de novo synthesis of [d(A-T)]-copolymer by preparations of E. coli DNA-polymerase I but do not effect primed synthesis ensured by this enzyme. High concentration of the substrate have similar effect. Using two-dimentional thin-layer chromatography and microcolumn chromatography on TEAE-cellulose we have shown that preliminary incubation of DNA-polymerase I preparations with dADP and dTDP results in the synthesis of short oligonucleotides (from di- to decanucleotides). Hydrolysis of these oligonucleotides with dilute sulfuric acid demonstrates that among the reaction products prevail oligoadenylates and oligothymidylates, but an appreciable amounts of heterooligomers including oligo[d(A-T)] were revealed as well. The model of so called de novo synthesis of regular polynucleotides is proposed, according to which dNDP-transferase, an accompanying enzyme in the preparations of DNA-polymerase I E. coli, is carrying out the synthesis of short oligonucleotides which form template-primer complexes repeatedly replicated by the DNA-polymerase I E. coli.

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