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首页> 外文期刊>Molecular and Cellular Biology >Initiation of simian virus 40 DNA replication in vitro: aphidicolin causes accumulation of early-replicating intermediates and allows determination of the initial direction of DNA synthesis.
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Initiation of simian virus 40 DNA replication in vitro: aphidicolin causes accumulation of early-replicating intermediates and allows determination of the initial direction of DNA synthesis.

机译：猿猴病毒40 DNA体外复制的启动：蚜虫菌素引起早期复制中间体的积累，并可以确定DNA合成的初始方向。

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Aphidicolin, a specific inhibitor of DNA polymerase alpha, provided a novel method for distinguishing between initiation of DNA synthesis at the simian virus 40 (SV40) origin of replication (ori) and continuation of replication beyond ori. In the presence of sufficient aphidicolin to inhibit total DNA synthesis by 50%, initiation of DNA replication in SV40 chromosomes or ori-containing plasmids continued in vitro, whereas DNA synthesis in the bulk of SV40 replicative intermediate DNA (RI) that had initiated replication in vivo was rapidly inhibited. This resulted in accumulation of early RI in which most nascent DNA was localized within a 600- to 700-base-pair region centered at ori. Accumulation of early RI was observed only under conditions that permitted initiation of SV40 ori-dependent, T-antigen-dependent DNA replication and only when aphidicolin was added to the in vitro system. Increasing aphidicolin concentrations revealed that DNA synthesis in the ori region was not completely resistant to aphidicolin but simply less sensitive than DNA synthesis at forks that were farther away. Since DNA synthesized in the presence of aphidicolin was concentrated in the 300 base pairs on the early gene side of ori, we conclude that the initial direction of DNA synthesis was the same as that of early mRNA synthesis, consistent with the model proposed by Hay and DePamphilis (Cell 28:767-779, 1982). The data were also consistent with initiation of the first DNA chains in ori by CV-1 cell DNA primase-DNA polymerase alpha. Synthesis of pppA/G(pN)6-8(pdN)21-23 chains on a single-stranded DNA template by a purified preparation of this enzyme was completely resistant to aphidicolin, and further incorporation of deoxynucleotide monophosphates was inhibited. Therefore, in the presence of aphidicolin, this enzyme could initiate RNA-primed DNA synthesis at ori first in the early gene direction and then in the late gene direction, but could not continue DNA synthesis for an extended distance.
机译：Aphidicolin是一种DNA聚合酶α的特异性抑制剂，它提供了一种新的方法来区分猿猴病毒40（SV40）复制起点（ori）的DNA合成起始和超越ori的复制继续。在有足够的蚜虫毒素抑制50％的总DNA合成的情况下，体外继续在SV40染色体或含ori的质粒中开始DNA复制，而在已经启动复制的大部分SV40复制中间DNA（RI）中进行DNA合成。体内被迅速抑制。这导致了早期RI的积累，其中大多数新生DNA定位在以ori为中心的600至700个碱基对的区域内。仅在允许启动SV40 ori依赖性，T抗原依赖性DNA复制的条件下，并且仅在将蚜虫碱添加到体外系统中时，才观察到早期RI的积累。蚜虫的浓度增加表明，ori区域的DNA合成不能完全抵抗蚜虫，但远不及叉处的DNA合成敏感。由于在蚜虫中存在的情况下合成的DNA集中在ori早期基因一侧的300个碱基对中，因此我们得出结论，DNA合成的初始方向与mRNA早期合成的方向相同，这与Hay和Han等人提出的模型一致。 DePamphilis（细胞28：767-779，1982）。数据也与CV-1细胞DNA primase-DNA聚合酶α起始的第一条DNA链一致。通过该酶的纯化制备，在单链DNA模板上合成pppA / G（pN）6-8（pdN）21-23链是完全抗蚜虫碱的，并且进一步抑制了脱氧核苷酸单磷酸的掺入。因此，在存在蚜虫的情况下，该酶可以先在ori的起始基因方向开始，然后再在基因的晚期起始RNA引发的DNA合成，但不能在更长的距离内继续DNA合成。

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