Drosophila engrailed-1,10-phenanthroline chimeras as probes of homeodomain-DNA complexes.
Drosophila engrailed-1,10-phenanthroline chimeras as probes of homeodomain-DNA complexes.
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果蝇将 1,10-菲咯啉嵌合体作为同源结构域-DNA 复合物的探针。
DOI：
10.1002/pro.5560041105
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发表时间：
1995
期刊：
影响因子：
--
通讯作者：
Sigman,DS
中科院分区：
文献类型：
--
作者：
Pan,CQ;Landgraf,R;Sigman,DS
我们通过将蛋白质与化学核酸酶 1,10-菲咯啉-铜 (OP-Cu) 连接，将果蝇 enrailed 同源结构域转化为序列特异性核酸酶。通过定点诱变将独特的半胱氨酸引入同源结构域的六个位置，以共价连接 OP-Cu。这些突变体不同的 DNA 结合亲和力和特异性以及它们的 OP-Cu 衍生物的 DNA 切割模式使我们能够评估嵌入的同源结构域-DNA 复合物的晶体结构。我们还利用一种同源域突变体 E28C 实现了位点特异性双链 DNA 断裂，该突变体有潜力用于识别基因组中的刻痕结合位点。由于同源结构域在含同源结构域的蛋白家族成员中非常保守，因此其他同源结构域蛋白可以通过在其同源结构域的 28 位连接 OP-Cu 来转化为核酸酶。
We have converted theDrosophilaengrailed homeodomain into a sequence‐specific nuclease by linking the protein to the chemical nuclease 1,10‐phenanthroline‐copper (OP‐Cu). Unique cysteines were introduced at six positions into the homeodomain by site‐directed mutagenesis for the covalent attachment of OP‐Cu. The varied DNA‐binding affinity and specificity of these mutants and the DNA cleavage pattern of their OP‐Cu derivatives allowed us to assess the crystal structure of the engrailed homeodomain‐DNA complex. We have also achieved site‐specific double‐stranded DNA scission with one of the homeodomain mutants, E28C, which has the potential of being used to identify engrailed binding sites in the genome. Because the homeodomain is so well conserved among members of the homeodomain‐containing protein family, other homeodomain proteins can be converted into nucleases by attaching OP‐Cu at position 28 of their homeodomains.