Human topoisomerase I promotes initiation of simian virus 40 DNA replication in vitro.
Human topoisomerase I promotes initiation of simian virus 40 DNA replication in vitro.
复制标题
人拓扑异构酶 I 促进猿猴病毒 40 DNA 体外复制的启动。
DOI：
10.1128/mcb.19.3.1686
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发表时间：
1999
影响因子：
5.3
通讯作者：
Simmons,DT
中科院分区：
文献类型：
--
作者：
Trowbridge,PW;Roy,R;Simmons,DT
将纯化的人拓扑异构酶 I (topo I) 添加到使用拓扑异构酶 I 缺陷提取物进行的猿猴病毒 40 T 抗原驱动的体外 DNA 复制反应中，可对完整分子产生超过 10 倍的刺激，并使整体 DNA 复制增强超过 3 倍。为了进一步表征这种刺激，我们首先证明牛 topo I 而不是大肠杆菌 I 也可以增强 DNA 复制。通过使用几种人类拓扑I突变体，我们表明需要催化活性形式的拓扑I。为了描述拓扑结构 I 是否影响复制的起始或延伸步骤，我们进行了延迟脉冲、脉冲追踪和延迟脉冲追踪实验。结果表明，topo I 不能促进部分复制分子的完成，但从反应一开始就需要它来启动复制。使用拓扑 I 结合 T 抗原片段 1-246T 和催化失活拓扑 I 突变体进行的竞争性抑制实验表明，拓扑 I 的部分复制刺激是通过与 T 抗原的直接相互作用介导的。总的来说，我们的数据表明，topo I 通过与 T 抗原形成必要的相互作用并刺激起始，从而增强完全复制 DNA 分子的合成。
Addition of purified human topoisomerase I (topo I) to simian virus 40 T antigen-driven in vitro DNA replication reactions performed with topo I-deficient extracts results in a greater than 10-fold stimulation of completed molecules as well as a more than 3-fold enhancement of overall DNA replication. To further characterize this stimulation, we first demonstrate that bovine topo I but notEscherichia colitopo I can also enhance DNA replication. By using several human topo I mutants, we show that a catalytically active form of topo I is required. To delineate whether topo I influences the initiation or the elongation step of replication, we performed delayed pulse, pulse-chase, and delayed pulse-chase experiments. The results illustrate that topo I cannot promote the completion of partially replicated molecules but is needed from the beginning of the reaction to initiate replication. Competitive inhibition experiments with the topo I binding T antigen fragment 1-246T and a catalytically inactive topo I mutant suggest that part of topo I’s stimulation of replication is mediated through a direct interaction with T antigen. Collectively, our data indicate that topo I enhances the synthesis of fully replicated DNA molecules by forming essential interactions with T antigen and stimulating initiation.