Maki S, Kornberg A
Department of Biochemistry, Stanford University School of Medicine, California 94305.
J Biol Chem. 1988 May 15;263(14):6547-54.
Escherichia coli dnaZX, the gene which when mutant blocks DNA chain elongation, was cloned into a lambda PL promoter-mediated expression vector. In cells carrying this plasmid, the activity that complements a mutant dnaZ extract in replicating a primed single-stranded DNA circle was increased about 20-fold. Two polypeptides of 71 and 52 kDa were overproduced. Upon fractionation, two complementing activities were purified to homogeneity and proved to be the 71- and 52-kDa polypeptides. Immunoassays revealed their respective identities with the tau and gamma subunits of DNA polymerase III holoenzyme. The N-terminal amino acid sequences of the first 12 residues were identical in both subunits, as were their molar specific activities in dnaZ complementation. Thus, the tau subunit complements the defect in the mutant holoenzyme from the dnaZts strain as efficiently as does the gamma subunit. Inasmuch as the 71-kDa subunit (tau) can also overcome the enzymatic defect in a dnaX mutant strain, this polypeptide has dual replication functions, only one of which can be performed by the gamma subunit. Availability of pure tau and gamma subunits for study has provided the basis for proposing an asymmetry in the structure and function of a dimeric DNA polymerase III holoenzyme.
大肠杆菌dnaZX基因（该基因发生突变时会阻断DNA链的延伸）被克隆到一个由λPL启动子介导的表达载体中。在携带该质粒的细胞中，在复制带引物的单链DNA环时补充突变型dnaZ提取物的活性增加了约20倍。产生了两种分子量分别为71 kDa和52 kDa的多肽。经过分级分离，两种互补活性被纯化至同质，并被证明是71 kDa和52 kDa的多肽。免疫分析揭示了它们分别与DNA聚合酶III全酶的τ和γ亚基相同。两个亚基前12个残基的N端氨基酸序列相同，它们在dnaZ互补中的摩尔比活性也相同。因此，τ亚基与γ亚基一样有效地弥补了来自dnaZts菌株的突变型全酶中的缺陷。由于71 kDa亚基（τ）也能克服dnaX突变菌株中的酶缺陷，该多肽具有双重复制功能，其中只有一种功能可由γ亚基执行。纯τ和γ亚基的可得性为研究提供了基础，据此提出二聚体DNA聚合酶III全酶在结构和功能上存在不对称性。
