Nethanel T, Zlotkin T, Kaufmann G
Department of Biochemistry, Tel Aviv University, Israel.
J Virol. 1992 Nov;66(11):6634-40. doi: 10.1128/JVI.66.11.6634-6640.1992.
We have previously proposed that DNA polymerase alpha-primase provides short RNA-DNA precursors below 40 nucleotides (DNA primers), several of which assemble into an Okazaki piece after intervening RNA has been removed and the gaps have been filled by DNA polymerase delta (or epsilon) (T. Nethanel, S. Reisfeld, G. Dinter-Gottlieb, and G. Kaufmann, J. Virol. 62:2867-2873, 1988; T. Nethanel and G. Kaufmann, J. Virol. 64:5912-5918, 1990). In this report, we confirm and extend these conclusions by studying the effects of deoxynucleoside triphosphate (dNTP) concentrations and the presence of ATP on the occurrence, dynamics, and configuration of DNA primers in simian virus 40 replicative intermediate DNA. We first show that these parameters are not significantly affected by a 10-fold increase in dNTP precursor concentrations. We then demonstrate that Okazaki piece synthesis can be arrested at the level of DNA primers by ATP depletion. The arrested DNA primers faced short gaps of 10 to 20 nucleotides at their 3' ends and were progressively chased into Okazaki pieces when ATP was restored. ATP could not be substituted in this process by adenosine-5'-O-(3-thiotriphosphate) or adenyl-imidodiphosphate. The chase was interrupted by aphidicolin but not by butylphenyl-dGTP. The results implicate an ATP-requiring factor in the switch between the two DNA polymerases engaged in Okazaki piece synthesis. They also suggest that the replication fork advances by small, DNA primer-size increments.
我们之前曾提出，DNA聚合酶α-引发酶可提供长度低于40个核苷酸的短RNA-DNA前体（DNA引物），在间隔RNA被去除且间隙由DNA聚合酶δ（或ε）填补后，其中几个引物会组装成冈崎片段（T. 内塔内尔、S. 赖斯费尔德、G. 丁特-戈特利布和G. 考夫曼，《病毒学杂志》62:2867 - 2873，1988；T. 内塔内尔和G. 考夫曼，《病毒学杂志》64:5912 - 5918，1990）。在本报告中，我们通过研究脱氧核苷三磷酸（dNTP）浓度和ATP的存在对猴病毒40复制中间体DNA中DNA引物的出现、动态变化及构象的影响，证实并扩展了这些结论。我们首先表明，dNTP前体浓度增加10倍对这些参数没有显著影响。然后我们证明，通过ATP消耗，冈崎片段的合成可在DNA引物水平被阻断。被阻断的DNA引物在其3'端面临10至20个核苷酸的短间隙，当恢复ATP时，它们会逐渐被转化为冈崎片段。在此过程中，ATP不能被腺苷 - 5'-O-(3 - 硫代三磷酸)或腺苷 - 亚氨二磷酸替代。这种转化被阿非科林阻断，但未被丁基苯基 - dGTP阻断。结果表明，在参与冈崎片段合成的两种DNA聚合酶之间的转换中涉及一个需要ATP的因子。它们还表明，复制叉以与DNA引物大小相当的小增量前进。
