Jazwinski S M
Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, New Orleans 70112.
Biochem J. 1987 Aug 15;246(1):213-9. doi: 10.1042/bj2460213.
The activity that replicates yeast DNA in vitro can be isolated from cells of the budding yeast Saccharomyces in a high-Mr (approximately 2 X 10(6] form. Several lines of evidence indicate that this fraction contains a multiprotein replicative complex. A functional assay has been developed for the analysis of the interaction of the replicating activity with DNA. Binding of the activity required Mg2+, but did not require the addition of ATP or the other ribo- or deoxynucleoside triphosphates. However, the ATP analogues adenosine 5'-[gamma-thio]triphosphate and adenosine 5'-[beta gamma-imido]triphosphate blocked the binding, suggesting that ATP participates in the interaction at some stage. The binding was template (origin)-specific in either the presence or the absence of ATP and the other nucleoside triphosphates; however, ATP stabilized the replicating activity. The preferential inhibition of binding that was observed in the presence of the DNA topoisomerase II inhibitor coumermycin suggests that the requirement for ATP may be at least partially accounted for by the involvement of this enzyme in the initial interaction of the replicating activity with DNA. Finally, the binding was rapid. In contrast, DNA synthesis displayed a lag when assayed directly without first allowing a period for the replicating activity to bind to the DNA. In addition, binding was 'tight', as judged by the resistance of the protein--DNA complexes to salt in comparison with the relative sensitivity of binding. The replicating activity was not readily displaced from the complexes by exogenous DNAs, either possessing or lacking yeast origins of replication. The results suggest that the interaction of the replicating activity with the DNA occurs in more than one stage.
体外复制酵母DNA的活性可以从出芽酵母酿酒酵母的细胞中以高分子量（约2×10⁶）的形式分离出来。几条证据表明，该组分含有一种多蛋白复制复合体。已开发出一种功能测定法来分析复制活性与DNA的相互作用。该活性的结合需要Mg²⁺，但不需要添加ATP或其他核糖或脱氧核苷三磷酸。然而，ATP类似物腺苷5'-[γ-硫代]三磷酸和腺苷5'-[βγ-亚氨基]三磷酸会阻断结合，这表明ATP在某个阶段参与了这种相互作用。无论是否存在ATP和其他核苷三磷酸，结合都是模板（起始位点）特异性的；然而，ATP能稳定复制活性。在DNA拓扑异构酶II抑制剂香豆霉素存在的情况下观察到的结合优先抑制现象表明，对ATP的需求可能至少部分是由于该酶参与了复制活性与DNA的初始相互作用。最后，结合很快。相比之下，直接测定DNA合成时会出现延迟，而没有先让复制活性与DNA结合一段时间。此外，从蛋白质-DNA复合体对盐的抗性与结合的相对敏感性判断，结合是“紧密的”。无论是具有还是缺乏酵母复制起始位点的外源DNA都不容易将复制活性从复合体中置换出来。结果表明，复制活性与DNA的相互作用分多个阶段进行。
