Applegren N, Hickey R J, Kleinschmidt A M, Zhou Q, Coll J, Wills P, Swaby R, Wei Y, Quan J Y, Lee M Y
Department of Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine, Baltimore, MD 21201, USA.
J Cell Biochem. 1995 Sep;59(1):91-107. doi: 10.1002/jcb.240590111.
Evidence for multiprotein complexes playing a role in DNA replication has been growing over the years. We have previously reported on a replication-competent multiprotein form of DNA polymerase isolated from human (HeLa) cell extracts. The proteins that were found at that time to co-purify with the human cell multiprotein form of DNA polymerase included: DNA polymerase alpha, DNA primase, topoisomerase I, RNase H, PCNA, and a DNA-dependent ATPase. The multiprotein form of the human cell DNA polymerase was further purified by Q-Sepharose chromatography followed by glycerol gradient sedimentation and was shown to be fully competent to support origin-specific and large T-antigen dependent simian virus 40 (SV40) DNA replication in vitro [Malkas et al. (1990b): Biochemistry 29:6362-6374]. In this report we describe the further characterization of the human cell replication-competent multiprotein form of DNA polymerase designated MRC. Several additional DNA replication proteins that co-purify with the MRC have been identified. These proteins include: DNA polymerase delta, RF-C, topoisomerase II, DNA ligase I, DNA helicase, and RP-A. The replication requirements, replication initiation kinetics, and the ability of the MRC to utilize minichromosome structures for DNA synthesis have been determined. We also report on the results of experiments to determine whether nucleotide metabolism enzymes co-purify with the human cell MRC. We recently proposed a model to represent the MRC that was isolated from murine cells [Wu et al. (1994): J Cell Biochem 54:32-46]. We can now extend this model to include the human cell MRC based on the fractionation, chromatographic and sedimentation behavior of the human cell DNA replication proteins. A full description of the model is discussed. Our experimental results provide further evidence to suggest that DNA synthesis is mediated by a multiprotein complex in mammalian cells.
多年来，关于多蛋白复合物在DNA复制中发挥作用的证据不断增加。我们之前报道过从人（HeLa）细胞提取物中分离出的具有复制能力的DNA聚合酶多蛋白形式。当时发现与人类细胞DNA聚合酶多蛋白形式共纯化的蛋白质包括：DNA聚合酶α、DNA引发酶、拓扑异构酶I、核糖核酸酶H、增殖细胞核抗原（PCNA）和一种依赖DNA的ATP酶。人类细胞DNA聚合酶的多蛋白形式通过Q-琼脂糖凝胶层析进一步纯化，随后进行甘油梯度沉降，并显示出在体外完全能够支持起源特异性和大T抗原依赖性猿猴病毒40（SV40）DNA复制[马尔卡斯等人（1990b）：《生物化学》29：6362 - 6374]。在本报告中，我们描述了对指定为MRC的人类细胞具有复制能力的DNA聚合酶多蛋白形式的进一步表征。已鉴定出几种与MRC共纯化的其他DNA复制蛋白。这些蛋白质包括：DNA聚合酶δ、复制因子C（RF-C）、拓扑异构酶II、DNA连接酶I、DNA解旋酶和复制蛋白A（RP-A）。已经确定了复制要求、复制起始动力学以及MRC利用微型染色体结构进行DNA合成的能力。我们还报告了确定核苷酸代谢酶是否与人类细胞MRC共纯化的实验结果。我们最近提出了一个模型来表示从鼠细胞中分离出的MRC[吴等人（1994）：《细胞生物化学杂志》54：32 - 46]。现在，基于人类细胞DNA复制蛋白的分级分离、色谱和沉降行为，我们可以将这个模型扩展到包括人类细胞MRC。对该模型进行了全面描述。我们的实验结果提供了进一步的证据，表明DNA合成是由哺乳动物细胞中的多蛋白复合物介导的。
