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首页> 外文期刊>Molecular and Cellular Biology >Simian virus 40 DNA replication in vitro: identification of multiple stages of initiation.
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Simian virus 40 DNA replication in vitro: identification of multiple stages of initiation.

机译：猿猴病毒40 DNA体外复制：鉴定多个起始阶段。

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A cell-free DNA replication system dependent upon five purified cellular proteins, one crude cellular fraction, and the simian virus 40 (SV40)-encoded large tumor antigen (T antigen) initiated and completed replication of plasmids containing the SV40 origin sequence. DNA synthesis initiated at or near the origin sequence after a time lag of approximately 10 min and then proceeded bidirectionally from the origin to yield covalently closed, monomer daughter molecules. The time lag could be completely eliminated by a preincubation of SV40 ori DNA in the presence of T antigen, a eucaryotic single-stranded DNA-binding protein (replication factor A [RF-A]), and topoisomerases I and II. In contrast, if T antigen and the template DNA were incubated alone, the time lag was only partially decreased. Kinetic analyses of origin recognition by T antigen, origin unwinding, and DNA synthesis suggest that the time lag in replication was due to the formation of a complex between T antigen and DNA called the T complex, followed by formation of a second complex called the unwound complex. Formation of the unwound complex required RF-A. When origin unwinding was coupled to DNA replication by the addition of a partially purified cellular fraction (IIA), DNA synthesis initiated at the ori sequence, but the template DNA was not completely replicated. Complete DNA replication in this system required the proliferating-cell nuclear antigen and another cellular replication factor, RF-C, during the elongation stage. In a less fractionated system, another cellular fraction, SSI, was previously shown to be necessary for reconstitution of DNA replication. The SSI fraction was required in the less purified system to antagonize the inhibitory action of another cellular protein(s). This inhibitor specifically blocked the earliest stage of DNA replication, but not the later stages. The implications of these results for the mechanisms of initiation and elongation of DNA replication are discussed.
机译：一个无细胞的DNA复制系统，该系统依赖于五种纯化的细胞蛋白，一种粗制的细胞级分和猿猴病毒40（SV40）编码的大肿瘤抗原（T抗原），从而启动并完成了包含SV40起源序列的质粒的复制。在大约10分钟的时间间隔后，DNA合成在起始序列或其附近开始，然后从起始方向双向进行，以产生共价封闭的单体子分子。通过在T抗原，真核单链DNA结合蛋白（复制因子A [RF-A]）和拓扑异构酶I和II存在下进行SV40 ori DNA的预孵育，可以完全消除时滞。相反，如果将T抗原和模板DNA单独孵育，则时滞只会部分减少。通过T抗原进行的起源识别，起源解开和DNA合成的动力学分析表明，复制的时间滞后是由于T抗原和DNA之间形成了称为T复合物的复合物，然后形成了称为解链的第二复合物复杂。展开复合物的形成需要RF-A。当通过添加部分纯化的细胞级分（IIA）将起点解链与DNA复制耦合时，DNA合成在ori序列处开始，但模板DNA没有完全复制。在该系统中，DNA的完全复制需要在延长阶段增殖细胞核抗原和另一个细胞复制因子RF-C。在分级程度较低的系统中，先前已证明另一种细胞级分SSI对于重组DNA复制是必需的。在较少纯化的系统中需要SSI馏分来拮抗另一种细胞蛋白的抑制作用。该抑制剂特异性地阻断了DNA复制的最早阶段，但没有阻止后期阶段。讨论了这些结果对DNA复制的起始和延伸机制的影响。

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