Gruz P, Matsui K, Sofuni T, Nohmi T
Division of Genetics and Mutagenesis, National Institute of Health Sciences, Tokyo, Japan.
Mutat Res. 1996 Jul 22;354(2):157-70. doi: 10.1016/0027-5107(96)00006-1.
The two distinct mucAB and samAB operons originally isolated from the plasmids of Salmonella typhimurium encode proteins engaged in induced mutagenesis. They represent two extreme cases among the so far characterized members of the enterobacterial umuDC family in respect to both the strength and the specificity of their effect. It is suggested that the MucA and SamA proteins are post-translationally processed to MucA' and SamA', respectively, which lack the N-terminal 25 amino acids and are the active species in mutagenesis. For the purpose of characterizing the individual activities of these proteins, we developed a new system for their SOS-independent separate and controllable expression in enterobacteria. Besides the matured forms of MucA', SamA' as well as MucB and SamB proteins we also expressed hybrid HisTag-MucA' and HisTag-SamA' proteins in which a synthetic 24 amino acid HisTag region replaces the natural 25 amino acid N-terminal leader present in the MucA and SamA precursors. In this study, we analyzed the effect of the mutagenesis proteins on the UV mutability of S. typhimurium YG5144. None of the proteins, if expressed alone, promoted UV mutagenesis. Different combinations of the proteins promoted mutagenesis to different extents in the order MucA' + MucB > SamA' + SamB > or = HisTag-MucA' + MucB > or = SamA' + MucB > MucA' + SamB > HisTag-SamA' + SamB. The mutagenesis enhancing potential of the combinations with MucB protein decreased as the expression of the proteins increased while the mutagenesis enhancing potential of the combinations with SamB protein increased together with the increase in the expression. The artificially expressed MucA' + MucB proteins were as active as their MucAB counterparts expressed from the plasmid pKM101 in promoting UV mutagenesis, but they were remarkably more efficient than their pKM101-born counterparts in promoting spontaneous mutagenesis. We conclude that the MucA'B and SamA'B proteins are partly interchangeable and the functionality of the resulting A' + B complex is largely dependent on the appropriate B-protein.
最初从鼠伤寒沙门氏菌质粒中分离出的两个不同的mucAB和samAB操纵子编码参与诱导诱变的蛋白质。就其作用的强度和特异性而言，它们代表了肠杆菌umuDC家族中迄今为止已鉴定成员中的两种极端情况。有人提出，MucA和SamA蛋白分别在翻译后加工成MucA'和SamA'，它们缺少N端的25个氨基酸，并且是诱变中的活性形式。为了表征这些蛋白质的个体活性，我们开发了一种新系统，用于它们在肠杆菌中不依赖SOS的单独和可控表达。除了成熟形式的MucA'、SamA'以及MucB和SamB蛋白外，我们还表达了杂交HisTag-MucA'和HisTag-SamA'蛋白，其中一个合成的24个氨基酸的HisTag区域取代了MucA和SamA前体中天然存在的25个氨基酸的N端前导序列。在本研究中，我们分析了诱变蛋白对鼠伤寒沙门氏菌YG5144紫外线诱变率的影响。如果单独表达，这些蛋白质均不能促进紫外线诱变。不同的蛋白质组合以MucA' + MucB > SamA' + SamB > 或 = HisTag-MucA' + MucB > 或 = SamA' + MucB > MucA' + SamB > HisTag-SamA' + SamB的顺序在不同程度上促进诱变作用。与MucB蛋白组合的诱变增强潜力随着蛋白质表达的增加而降低，而与SamB蛋白组合的诱变增强潜力则随着表达的增加而增加。人工表达的MucA' + MucB蛋白在促进紫外线诱变方面与从质粒pKM101表达的MucAB对应物一样活跃，但在促进自发诱变方面比其源自pKM101的对应物明显更有效。我们得出结论，MucA'B和SamA'B蛋白部分可互换，并且所得A' + B复合物的功能很大程度上取决于合适的B蛋白。
