Kim K, Harshey R M
Department of Microbiology, University of Texas at Austin 78712, USA.
Nucleic Acids Res. 1995 Oct 11;23(19):3937-43. doi: 10.1093/nar/23.19.3937.
The transposase (A protein) of phage Mu encodes binding to two families of DNA sites, att sites located at the Mu ends and enhancer sites located internally. Separate subdomains in the N-terminal domain I of Mu A protein are known to be involved in recognition of the att and enhancer sites. We have delineated an approximately 135 aa region within domain I beta gamma that specifies binding to Mu att sites. This peptide was overexpressed and its properties compared with that of the larger domain I beta gamma as well as the intact Mu A protein. Extensive mutagenesis of residues around a putative helix-turn-helix DNA-binding motif within the I beta domain identified several mutants defective in DNA transposition in vivo. Of these, Mu A(K157Q) was completely defective in att DNA-binding. Mu A(F131S) and Mu A(R146N) had a lower affinity for att DNA and low levels of transposition in vitro. Our results indicate that residues in the gamma region are required for activity and that residues outside the beta gamma region must also influence discrimination between the multiple att sites.
噬菌体Mu的转座酶（A蛋白）编码与两类DNA位点结合，位于Mu两端的附着位点（att位点）和位于内部的增强子位点。已知Mu A蛋白N端结构域I中的不同亚结构域参与对att位点和增强子位点的识别。我们在结构域Iβγ内划定了一个约135个氨基酸的区域，该区域决定与Mu att位点的结合。该肽段被过量表达，并将其性质与较大的结构域Iβγ以及完整的Mu A蛋白的性质进行比较。对Iβ结构域内一个假定的螺旋-转角-螺旋DNA结合基序周围的残基进行广泛诱变，鉴定出几个在体内DNA转座中有缺陷的突变体。其中，Mu A(K157Q)在att DNA结合方面完全有缺陷。Mu A(F131S)和Mu A(R146N)对att DNA的亲和力较低，且在体外转座水平较低。我们的结果表明，γ区域中的残基是活性所必需的，并且βγ区域之外的残基也必定影响对多个att位点的区分。
