Thomas D C, Levy M, Sancar A
J Biol Chem. 1985 Aug 15;260(17):9875-83.
The UvrA, UvrB, and UvrC proteins of Escherichia coli are subunits of a DNA repair enzyme, ABC exci nuclease. In order to amplify these proteins, we have joined the artificial canonical promoter tac (Amann E., Brosius, J., and Ptashne, M. (1983) Gene (Amst.) 25, 167-178) to the uvr genes to obtain plasmids that express these genes under the control of the lac repressor. When cells carrying the tac-uvr plasmids are induced by the gratuitous lac inducer isopropyl-beta-D-galactoside the Uvr proteins are overproduced reaching a level of 10-20% of total cellular proteins after 6-8 h of induction. We have developed methods to purify all three Uvr proteins, UvrA, UvrB, and UvrC, in milligram quantities and to near homogeneity from these overproducing cells. The purified UvrA protein is an ATPase but UvrB and UvrC proteins are not. However, UvrB protein stimulates the ATPase activity of UvrA protein by a factor of 1.5 in the presence of double-stranded DNA and by a factor of about 2.6 in the presence of UV-irradiated DNA but not in the absence of DNA.
大肠杆菌的UvrA、UvrB和UvrC蛋白是一种DNA修复酶即ABC外切核酸酶的亚基。为了扩增这些蛋白，我们将人工合成的标准启动子tac（阿曼E.、布罗修斯J.和普塔什内M.（1983年），《基因》（阿姆斯特丹）25卷，第167 - 178页）与uvr基因连接，以获得在乳糖阻遏物控制下表达这些基因的质粒。当携带tac - uvr质粒的细胞被 gratuitous乳糖诱导剂异丙基 - β - D - 半乳糖苷诱导时，Uvr蛋白会过量产生，诱导6至8小时后达到细胞总蛋白的10% - 20%。我们已经开发出从这些过量产生蛋白的细胞中以毫克量纯化所有三种Uvr蛋白（UvrA、UvrB和UvrC）并使其接近均一性的方法。纯化后的UvrA蛋白是一种ATP酶，但UvrB和UvrC蛋白不是。然而，在双链DNA存在的情况下，UvrB蛋白会使UvrA蛋白的ATP酶活性提高1.5倍，在紫外线照射的DNA存在的情况下提高约2.6倍，但在没有DNA的情况下则不会。
