Su R T, DePamphilis M L
J Virol. 1978 Oct;28(1):53-65. doi: 10.1128/JVI.28.1.53-65.1978.
Three subnuclear systems capable of continuing many aspects of simian virus 40 (SV40) DNA replication were characterized in an effort to define the minimum requirements for "normal" DNA replication in vitro. Nuclear extracts, prepared by incubating nuclei isolated from SV40-infected CV-1 cells in a hypotonic buffer to release both SV40 replicating and mature chromosomes, were either centrifuged to separate the total SV40 nucleoprotein complexes from the soluble nucleosol or fractionated on sucrose gradients to provide purified SV40 replicating chromosomes. With nuclear extracts, CV-1 cell cytosol stimulated total DNA synthesis, elongation of nascent DNA chains, maturation and joining of "Okazaki pieces," and the conversion of replicating viral DNA into covalently closed, superhelical DNA. Nucleoprotein complexes responded similarly, but frequently the response was reduced by 10 to 30%. In contrast, isolated replicating chromosomes in the presence of cytosol appeared only to complete and join Okazaki pieces already present on the template; without cytosol, Okazaki pieces incorporated alpha-(32)P-labeled deoxynucleoside triphosphates but failed to join. Consequently, replicating chromosomes failed to extensively continue nascent DNA chain growth, and the conversion of viral replicating DNA into mature DNA was seven to eight times less than that observed in nuclear extracts. Addition of neither cytosol nor nucleosol corrected this problem. In the presence of cytosol, nonspecific endonuclease activity was not a problem in any of the three in vitro systems. Extensive purification of replicating chromosomes was limited by three as yet irreversible phenomena. First, replicating chromosomes isolated in a low-ionic-strength medium had a limited capability to continue DNA synthesis. Second, diluting either nuclear extracts or replicating chromosomes before incubation in vitro stimulated total DNA synthesis but was accompanied by the simultaneous appearance of small-molecular-weight nascent DNA not associated with intact viral DNA templates and a decrease in the synthesis of covalently closed viral DNA. Although this second phenomenon appeared similar to the first, template concentration alone could not account for the failure of purified replicating chromosomes to yield covalently closed DNA. Finally, preparation of nucleoprotein complexes in increasing concentrations of NaCl progressively decreased their ability to continue DNA replication. Exposure to 0.3 M NaCl removed one or more factors required for DNA synthesis which could be replaced by addition of cytosol. However, higher NaCl concentrations yielded nucleoprotein complexes that had relatively no endogenous DNA synthesis activity and that no longer responded to cytosol. These data demonstrate that continuation of endogenous DNA replication in vitro requires both the soluble cytosol fraction and a complex nucleoprotein template whose ability to continue DNA synthesis depends on its concentration and ionic environment during its preparation.
为了确定体外“正常”DNA复制的最低要求，对能够持续进行猿猴病毒40（SV40）DNA复制多个方面的三个亚核系统进行了表征。通过在低渗缓冲液中孵育从感染SV40的CV-1细胞中分离的细胞核来制备核提取物，以释放SV40复制染色体和成熟染色体，然后将其离心以从可溶性核溶胶中分离出总SV40核蛋白复合物，或在蔗糖梯度上分级分离以提供纯化的SV40复制染色体。使用核提取物时，CV-1细胞胞质溶胶可刺激总DNA合成、新生DNA链的延伸、“冈崎片段”的成熟和连接，以及将复制的病毒DNA转化为共价闭合的超螺旋DNA。核蛋白复合物的反应类似，但反应通常会降低10%至30%。相比之下，在胞质溶胶存在下分离的复制染色体似乎仅完成并连接模板上已有的冈崎片段；没有胞质溶胶时，冈崎片段掺入α-(32)P标记的脱氧核苷三磷酸，但无法连接。因此，复制染色体无法广泛地继续新生DNA链的生长，病毒复制DNA向成熟DNA的转化比在核提取物中观察到的少七至八倍。添加胞质溶胶或核溶胶均无法纠正此问题。在胞质溶胶存在下，非特异性核酸内切酶活性在任何一个体外系统中都不是问题。复制染色体的广泛纯化受到三种尚未可逆的现象的限制。首先，在低离子强度培养基中分离的复制染色体继续DNA合成的能力有限。其次，在体外孵育前稀释核提取物或复制染色体可刺激总DNA合成，但同时会出现与完整病毒DNA模板无关的小分子新生DNA，并且共价闭合病毒DNA的合成会减少。尽管第二种现象与第一种现象相似，但仅模板浓度无法解释纯化的复制染色体无法产生共价闭合DNA的原因。最后，在不断增加的NaCl浓度下制备核蛋白复合物会逐渐降低其继续DNA复制的能力。暴露于0.3 M NaCl会去除DNA合成所需的一种或多种因子，这些因子可通过添加胞质溶胶来替代。然而，更高的NaCl浓度会产生相对没有内源性DNA合成活性且不再对胞质溶胶作出反应的核蛋白复合物。这些数据表明，体外内源性DNA复制的持续需要可溶性胞质溶胶部分和复杂的核蛋白模板，其继续DNA合成的能力取决于其制备过程中的浓度和离子环境。
