Nazarenko I A, Bobko L E, Romashchenko A G, Khripin Iu L, Salganik R I
Mol Biol (Mosk). 1979 Jan-Feb;13(1):216-27.
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.
研究了由大肠杆菌DNA聚合酶I制剂催化的聚[d（A-T）]从头合成的初始事件。数据提供了证据表明脱氧核苷二磷酸：寡核苷酸脱氧核苷酸转移酶（dNDP转移酶），即能够催化dNDP的无引物聚合的酶，参与了起始过程。这一结论基于以下数据：1）用dADP和dTDT对大肠杆菌DNA聚合酶I制剂进行预孵育消除了聚[d（A-T）]合成中的滞后阶段；2）dithiothreitol和N-乙基马来酰亚胺，dNDP转移酶的抑制剂，抑制大肠杆菌DNA聚合酶I制剂对[d（A-T）]共聚物的从头合成，但不影响该酶确保的引物合成。高浓度的底物有类似的作用。使用二维薄层色谱和TEAE-纤维素微柱色谱，我们已经表明用dADP和dTDP对DNA聚合酶I制剂进行预孵育会导致短寡核苷酸（从二聚体到十聚体）的合成。用稀硫酸水解这些寡核苷酸表明，在反应产物中主要是寡腺苷酸和寡胸苷酸，但也发现了相当数量的包括寡聚[d（A-T）]在内的杂合寡聚物。提出了所谓的规则多核苷酸从头合成模型，根据该模型，dNDP转移酶，大肠杆菌DNA聚合酶I制剂中的一种伴随酶，正在进行短寡核苷酸的合成，这些短寡核苷酸形成由大肠杆菌DNA聚合酶I反复复制的模板-引物复合物。
