O'Donnell M, Studwell P S
Microbiology Department, Hearst Microbiology Research Center, Cornell University Medical College, New York, New York 10021.
J Biol Chem. 1990 Jan 15;265(2):1179-87.
DNA polymerase III holoenzyme (holoenzyme) is the 10-subunit replicase of the Escherichia coli chromosome. In this report, pure preparations of delta, delta', and a gamma chi psi complex are resolved from the five protein gamma complex subassembly. Using these subunits and other holoenzyme subunits isolated from overproducing plasmid strains of E. coli, the rapid and highly processive holoenzyme has been reconstituted from only five pure single subunits: alpha, epsilon, gamma, delta, and beta. The preceding report showed that of the three subunits in the core polymerase, only a complex of alpha (DNA polymerase) and epsilon (3'-5' exonuclease) are required to assemble a processive holoenzyme on a template containing a preinitiation complex (Studwell, P.S., and O'Donnell, M. (1990) J. Biol. Chem. 265, 1171-1178). This report shows that of the five proteins in the gamma complex only a heterodimer of gamma and delta is required with the beta subunit to form the ATP-activated preinitiation complex with a primed template. Surprisingly, the delta' subunit does not form an active complex with gamma but forms a fully active heterodimer complex with the tau subunit (as does delta). Hence, the tau delta' and gamma delta heterodimers are fully active in the preinitiation complex reaction with beta and primed DNA. Holoenzymes reconstituted using the alpha epsilon complex, beta subunit, and either gamma delta or tau delta' are fully processive in DNA synthesis, and upon completing the template they rapidly cycle to a new primed template endowed with a preinitiation complex clamp. Since the holoenzyme molecule contains all of these accessory subunits (gamma, delta, tau, delta', and beta) in all likelihood it has the capacity to form two preinitiation complex clamps simultaneously at two primer termini. Two primer binding components within one holoenzyme may mediate its rapid cycling to multiple primers on the lagging strand and also provides functional evidence for the hypothesis of holoenzyme as a dimeric polymerase capable of simultaneous replication of both leading and lagging strands of a replication fork.
DNA聚合酶III全酶（全酶）是大肠杆菌染色体的10亚基复制酶。在本报告中，从五个蛋白质γ复合体亚组件中解析出了δ、δ'和γχψ复合体的纯制剂。利用这些亚基以及从大肠杆菌过量生产质粒菌株中分离出的其他全酶亚基，仅从五个纯单亚基：α、ε、γ、δ和β就成功重构了快速且高度持续合成的全酶。之前的报告表明，在核心聚合酶的三个亚基中，只有α（DNA聚合酶）和ε（3'-5'核酸外切酶）的复合体需要在含有预引发复合体的模板上组装持续合成的全酶（Studwell, P.S., and O'Donnell, M. (1990) J. Biol. Chem. 265, 1171 - 1178）。本报告表明，在γ复合体的五个蛋白质中，只有γ和δ的异二聚体与β亚基一起才能与带引物的模板形成ATP激活的预引发复合体。令人惊讶的是，δ'亚基不与γ形成活性复合体，而是与τ亚基形成完全活性的异二聚体复合体（δ也是如此）。因此，τδ'和γδ异二聚体在与β和带引物的DNA的预引发复合体反应中完全有活性。使用αε复合体、β亚基以及γδ或τδ'重构的全酶在DNA合成中具有完全的持续合成能力，并且在完成模板后，它们会迅速循环到一个带有预引发复合体夹子的新的带引物模板上。由于全酶分子可能含有所有这些辅助亚基（γ、δ、τ、δ'和β），它很有可能有能力在两个引物末端同时形成两个预引发复合体夹子。一个全酶内的两个引物结合组件可能介导其快速循环到滞后链上的多个引物，也为全酶作为一种能够同时复制复制叉的前导链和滞后链的二聚体聚合酶这一假说提供了功能证据。
