Wu Z, Chaconas G
Department of Biochemistry, University of Western Ontario, London, Canada.
EMBO J. 1995 Aug 1;14(15):3835-43. doi: 10.1002/j.1460-2075.1995.tb00053.x.
The Mu A protein is a 75 kDa transposase organized into three structural domains. By severing the C-terminal region (domain III) from the remainder of the protein, we unmasked a novel non-specific DNA binding and nuclease activity in this region. Deletion analysis localized both activities to a 26 amino acid stretch (aa 575-600) which remarkably remained active in DNA binding and cleavage. The two activities were shown to be tightly linked by site-directed mutagenesis. To study the importance of these activities in the transposition process, an intact mutant transposase lacking the DNA binding and nuclease activity of domain III was constructed and purified. The mutant transposase was indistinguishable from wild-type Mu A in binding affinity for both the Mu ends and the enhancer, and in strand transfer activity when the cleavage step was bypassed. In contrast, the mutant transposase displayed defects in both synapsis and donor cleavage. Our results strongly suggest that the 26 amino acid region in domain III carries catalytic residues required for donor DNA cleavage by Mu A protein. Furthermore, our data suggest that an active site for donor cleavage activity in the Mu tetramer is assembled from domain II (metal ion binding) in one A monomer and domain III (DNA cleavage) in a separate A monomer. This proposal for active site assembly is in agreement with the recently proposed domain sharing model by Yang et al. (Yang, J.Y., Kim, K., Jayaram, M. and Harshey, R.M. [1995] EMBO J., 14, 2374-2384).
Mu A蛋白是一种75 kDa的转座酶，由三个结构域组成。通过将C末端区域（结构域III）与蛋白质的其余部分切断，我们在该区域揭示了一种新的非特异性DNA结合和核酸酶活性。缺失分析将这两种活性定位到一个26个氨基酸的片段（第575 - 600位氨基酸），该片段在DNA结合和切割方面仍具有显著活性。通过定点诱变表明这两种活性紧密相连。为了研究这些活性在转座过程中的重要性，构建并纯化了一种完整的突变转座酶，该酶缺乏结构域III的DNA结合和核酸酶活性。该突变转座酶在对Mu末端和增强子的结合亲和力以及绕过切割步骤时的链转移活性方面与野生型Mu A没有区别。相比之下，突变转座酶在突触形成和供体切割方面都表现出缺陷。我们的结果强烈表明，结构域III中的26个氨基酸区域携带了Mu A蛋白切割供体DNA所需的催化残基。此外，我们的数据表明，Mu四聚体中供体切割活性的活性位点是由一个A单体中的结构域II（金属离子结合）和另一个单独的A单体中的结构域III（DNA切割）组装而成。这种活性位点组装的提议与Yang等人最近提出的结构域共享模型一致（Yang, J.Y., Kim, K., Jayaram, M.和Harshey, R.M. [1995] EMBO J., 14, 2374 - 2384）。
