Delagoutte E, von Hippel P H
Institute of Molecular Biology and Department of Chemistry, University of Oregon, Eugene, Oregon 97403, USA.
Biochemistry. 2001 Apr 10;40(14):4459-77. doi: 10.1021/bi001306l.
Processive strand-displacement DNA synthesis with the T4 replication system requires functional "coupling" between the DNA polymerase (gp43) and the helicase (gp41). To define the physical basis of this functional coupling, we have used analytical ultracentrifugation to show that gp43 is a monomeric species at physiological protein concentrations and that gp41 and gp43 do not physically interact in the absence of DNA, suggesting that the functional coupling between gp41 and gp43 depends significantly on interactions modulated by the replication fork DNA. Results from strand-displacement DNA synthesis show that a minimal gp41-gp43 replication complex can perform strand-displacement synthesis at approximately 90 nts/s in a solution containing poly(ethylene glycol) to drive helicase loading. In contrast, neither the Klenow fragment of Escherichia coli DNA polymerase I nor the T7 DNA polymerase, both of which are nonprocessive polymerases, can carry out strand-displacement DNA synthesis with gp41, suggesting that the functional helicase-polymerase coupling may require the homologous system. However, we show that a heterologous helicase-polymerase pair can work if the polymerase is processive. Strand-displacement DNA synthesis using the gp41 helicase with the T4 DNA polymerase holoenzyme or the phage T7 DNA polymerase-thioredoxin complex, both of which are processive, proceeds at the rate of approximately 250 nts/s. However, replication fork assembly is less efficient with the heterologous helicase-polymerase pair. Therefore, a processive (homologous or heterologous) "trailing" DNA polymerase is sufficient to improve gp41 processivity and unwinding activity in the elongation stage of the helicase reaction, and specific T4 helicase-polymerase coupling becomes significant only in the assembly (or initiation) stage.
利用T4复制系统进行的连续性链置换DNA合成需要DNA聚合酶（gp43）和解旋酶（gp41）之间具有功能性“偶联”。为了确定这种功能性偶联的物理基础，我们使用分析超速离心法表明，在生理蛋白质浓度下，gp43是一种单体，并且在没有DNA的情况下，gp41和gp43不会发生物理相互作用，这表明gp41和gp43之间的功能性偶联在很大程度上取决于由复制叉DNA调节的相互作用。链置换DNA合成的结果表明，一个最小的gp41-gp43复制复合物在含有聚乙二醇以驱动解旋酶装载的溶液中，能够以大约90个核苷酸/秒的速度进行链置换合成。相比之下，大肠杆菌DNA聚合酶I的Klenow片段和T7 DNA聚合酶这两种非连续性聚合酶，都不能与gp41一起进行链置换DNA合成，这表明功能性解旋酶-聚合酶偶联可能需要同源系统。然而，我们表明，如果聚合酶是连续性的，异源解旋酶-聚合酶对也可以发挥作用。使用gp41解旋酶与T4 DNA聚合酶全酶或噬菌体T7 DNA聚合酶-硫氧还蛋白复合物（两者都是连续性的）进行链置换DNA合成，其速度约为250个核苷酸/秒。然而，异源解旋酶-聚合酶对的复制叉组装效率较低。因此，一个连续性的（同源或异源）“尾随”DNA聚合酶足以在解旋酶反应的延伸阶段提高gp41的连续性和解旋活性，并且特定的T4解旋酶-聚合酶偶联仅在组装（或起始）阶段才变得重要。
