Maki H, Maki S, Kornberg A
Department of Biochemistry, Stanford University School of Medicine, California 94305.
J Biol Chem. 1988 May 15;263(14):6570-8.
Pol III, a subassembly of Escherichia coli DNA polymerase III holoenzyme lacking only the auxiliary beta subunit, was purified to homogeneity by an improved procedure. This assembly consists of nine different polypeptides, likely in a 1:1 stoichiometry: a catalytic core (pol III) of alpha (132 kDa), epsilon (27 kDa), and theta (10 kDa), and six auxiliary subunits: tau (71 kDa), gamma (52 kDa), delta (35 kDa), delta' (33 kDa), chi (15 kDa), and psi (12 kDa). The assembly behaves on gel filtration as a particle of about 800 kDa, indicating a content of two each of the subunits. A new procedure for purifying the core yielded a novel dimeric form which may provide the foundation for the dimeric nature of the more complex pol III and holoenzyme forms. Pol III readily dissociates into several subassemblies including pol III', likely a dimeric core with two tau subunits. The holoenzyme, purified by a similar procedure with ATP and Mg2+ present throughout, retained the beta subunit (37 kDa) as well as all the subunits present in pol III; the mass of the holoenzyme was estimated to be 900 kDa. The isolated initiation complex of holoenzyme with a primed template DNA and the elongation complex (formed in the presence of three deoxynucleoside triphosphates) had the same composition and stoichiometry as observed for pol III with two beta dimers in addition. An initiation complex assembled from a mixture of monomeric pol III core, gamma 2 delta delta' chi psi complex (gamma complex), beta, and tau retained the core, one beta dimer, and two tau subunits but was deficient in the gamma complex. When tau was omitted from the assembly mixture, the initiation complex contained one or two gamma complexes instead of the tau subunit. Based on these data, pol III holoenzyme is judged to be an asymmetric dimeric particle with twin pol III core active sites and two different sets of auxiliary units designed to achieve essentially concurrent replication of both leading and lagging strand templates.
大肠杆菌DNA聚合酶III全酶的一个亚组件Pol III，仅缺少辅助性β亚基，通过一种改进的方法被纯化至同质。该组件由九种不同的多肽组成，可能以1:1的化学计量比存在：一个催化核心（pol III），由α（132 kDa）、ε（27 kDa）和θ（10 kDa）组成，以及六个辅助亚基：τ（71 kDa）、γ（52 kDa）、δ（35 kDa）、δ'（33 kDa）、χ（15 kDa）和ψ（12 kDa）。该组件在凝胶过滤中表现为约800 kDa的颗粒，表明每个亚基含有两个。一种纯化核心的新方法产生了一种新的二聚体形式，这可能为更复杂的pol III和全酶形式的二聚体性质提供基础。Pol III很容易解离成几个亚组件，包括pol III'，可能是一个带有两个τ亚基的二聚体核心。全酶通过类似的方法在整个过程中加入ATP和Mg2+进行纯化，保留了β亚基（37 kDa）以及pol III中存在的所有亚基；全酶的质量估计为900 kDa。分离出的全酶与带引物的模板DNA的起始复合物和延伸复合物（在三种脱氧核苷三磷酸存在下形成）具有与pol III相同的组成和化学计量比，此外还含有两个β二聚体。由单体pol III核心、γ2δδ'χψ复合物（γ复合物）、β和τ的混合物组装而成的起始复合物保留了核心、一个β二聚体和两个τ亚基，但缺乏γ复合物。当从组装混合物中省略τ时，起始复合物包含一个或两个γ复合物而不是τ亚基。基于这些数据，pol III全酶被判定为一个不对称的二聚体颗粒，具有双pol III核心活性位点和两组不同的辅助单元，旨在实现前导链和滞后链模板的基本同时复制。
