Mutational analysis of the att DNA-binding domain of phage Mu transposase.
Mutational analysis of the att DNA-binding domain of phage Mu transposase.
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噬菌体 Mu 转座酶 att DNA 结合域的突变分析。
DOI：
10.1093/nar/23.19.3937
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发表时间：
1995
影响因子：
14.9
通讯作者：
Harshey,RM
中科院分区：
文献类型：
--
作者：
Kim,K;Harshey,RM
噬菌体 Mu 的转座酶（A 蛋白）编码与两个 DNA 位点家族的结合，即位于 Mu 末端的 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 中的单独子结构域参与对附加子和增强子位点的识别。我们在 1βγ 域内划定了约 135 个氨基酸区域，该区域指定与 Muattsites 的结合。该肽被过表达，其特性与较大结构域 1βγ 以及完整的 Mu A 蛋白相比。 1βγβ结构域内推定的螺旋-转角-螺旋DNA结合基序周围残基的广泛突变发生鉴定了几种体内DNA转座缺陷的突变体。其中，Mu A(K157Q) 在attDNA 结合方面完全有缺陷。 Mu A(F131S)和Mu A(R146N)对attDNA的亲和力较低，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须影响多个 att 位点之间的区分。
The transposase (A protein) of phage Mu encodes binding to two families of DNA sites,attsites 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 theattand enhancer sites. We have delineated an ˜135 aa region within domain 1βγ that specifies binding to Muattsites. This peptide was overexpressed and its properties compared with that of the larger domain 1βγ as well as the intact Mu A protein. Extensive muta genesis of residues around a putative helix-turn-helix DNA-binding motif within the 1βγβ domain identified several mutants defective in DNA transpositionin vivo. Of these, Mu A(K157Q) was completely defective inattDNA-binding. Mu A(F131S) and Mu A(R146N) had a lower affinity forattDNA and low levels of transpositionin vitro. Our results indicate that residues in the γ region are required for activity and that residues outside the βγ region must also influence discrimination between the multiple att sites.