Coll J M, Sekowski J W, Hickey R J, Schnaper L, Yue W, Brodie A M, Uitto L, Syvaoja J E, Malkas L H
Department of Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine, Baltimore 21201, USA.
Oncol Res. 1996;8(10-11):435-47.
In this report, we describe for the first time the isolation and purification of a multiprotein complex for DNA replication from MDA MB-468 human breast cancer cells. This complex, which we designate the DNA synthesome, fully supports the in vitro replication of simian virus 40 (SV40) origin-containing DNA in the presence of the viral large T-antigen. Since the SV40 virus utilizes the host's cellular proteins for its own DNA replication, our results indicate that the DNA synthesome may play a role not only in viral DNA synthesis but in human breast cell DNA replication as well. Our studies demonstrate that the following DNA replication proteins constitute the DNA synthesome: DNA polymerase alpha, DNA primase, DNA polymerase delta, proliferating cell nuclear antigen, replication protein A, replication factor C, DNA topoisomerases I, II, and DNA polymerase epsilon. In addition, we successfully isolated the DNA synthesome from human breast tumor tissue as well as from xenografts from nude mice injected with the human breast cancer cell line MCF-7. The DNA synthesome purified from the breast cancer tissues fully supports SV40 DNA replication in vitro. Furthermore, our results obtained from a novel forward mutagenesis assay suggest that the DNA synthesome isolated from a nonmalignant breast cell line mediates SV40 DNA replication by an error-resistant mechanism. In contrast, the DNA synthesome derived from malignant breast cells and tissue exhibited a lower fidelity for DNA synthesis in vitro. Overall, our data support the role of the DNA synthesome as mediating breast cell DNA replication in vitro and in vivo.
在本报告中，我们首次描述了从MDA MB - 468人乳腺癌细胞中分离和纯化用于DNA复制的多蛋白复合物。我们将这种复合物命名为DNA合成体，在病毒大T抗原存在的情况下，它能完全支持含猿猴病毒40（SV40）原点的DNA的体外复制。由于SV40病毒利用宿主细胞蛋白进行自身DNA复制，我们的结果表明DNA合成体可能不仅在病毒DNA合成中起作用，在人乳腺细胞DNA复制中也起作用。我们的研究表明，以下DNA复制蛋白构成了DNA合成体：DNA聚合酶α、DNA引发酶、DNA聚合酶δ、增殖细胞核抗原、复制蛋白A、复制因子C、DNA拓扑异构酶I、II以及DNA聚合酶ε。此外，我们成功地从人乳腺肿瘤组织以及注射了人乳腺癌细胞系MCF - 7的裸鼠异种移植物中分离出了DNA合成体。从乳腺癌组织中纯化的DNA合成体在体外能完全支持SV40 DNA复制。此外，我们从一种新型正向诱变试验中获得的结果表明，从非恶性乳腺细胞系中分离出的DNA合成体通过一种抗错机制介导SV40 DNA复制。相比之下，源自恶性乳腺细胞和组织的DNA合成体在体外DNA合成中表现出较低的保真度。总体而言，我们的数据支持DNA合成体在体外和体内介导乳腺细胞DNA复制的作用。
