Conversion of a helix-turn-helix motif sequence-specific DNA binding protein into a site-specific DNA cleavage agent.
Conversion of a helix-turn-helix motif sequence-specific DNA binding protein into a site-specific DNA cleavage agent.
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将螺旋-转角-螺旋基序序列特异性 DNA 结合蛋白转化为位点特异性 DNA 切割剂。
DOI：
10.1073/pnas.87.8.2882
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发表时间：
1990
影响因子：
11.1
通讯作者：
Gunasekera,A
中科院分区：
文献类型：
--
作者：
Ebright,RH;Ebright,YW;Pendergrast,PS;Gunasekera,A
大肠杆菌分解代谢物基因激活蛋白（CAP）是螺旋-转角-螺旋基序序列特异性DNA结合蛋白[de Crombrugghe，B.，Busby，S. &布克，H.（1984）Science 224，831-838;和Pabo，C. & Sauer，R.（1984）Annu. Rev.Biochem.53，293-321]。在这项工作中，CAP已被转化为一个位点特异性的DNA切割剂的螯合剂1，10-菲咯啉在螺旋-转角-螺旋基序的氨基酸10的掺入。[（N-乙酰基-5-氨基-1，10-菲咯啉）-Cys 178]CAP结合22个碱基对的DNA识别位点，Kobs = 1 x 10（8）M-1。在Cu（II）和还原剂存在下，[（N-乙酰基-5-氨基-1，10-菲咯啉）-Cys 178]CAP在DNA识别位点内的每个DNA链上的四个相邻核苷酸处切割DNA。已使用40碱基对和7164碱基对DNA底物证明了DNA切割反应。DNA切割反应不受DNA底物的dam甲基化的抑制。这种半合成的位点特异性DNA切割剂在染色体作图、克隆和测序中具有潜在的应用。
Escherichia coli catabolite gene activator protein (CAP) is a helix-turn-helix motif sequence-specific DNA binding protein [de Crombrugghe, B., Busby, S. & Buc, H. (1984) Science 224, 831-838; and Pabo, C. & Sauer, R. (1984) Annu. Rev. Biochem. 53, 293-321]. In this work, CAP has been converted into a site-specific DNA cleavage agent by incorporation of the chelator 1,10-phenanthroline at amino acid 10 of the helix-turn-helix motif. [(N-Acetyl-5-amino-1,10-phenanthroline)-Cys178]CAP binds to a 22-base-pair DNA recognition site with Kobs = 1 x 10(8) M-1. In the presence of Cu(II) and reducing agent, [(N-acetyl-5-amino-1,10-phenanthroline)-Cys178]CAP cleaves DNA at four adjacent nucleotides on each DNA strand within the DNA recognition site. The DNA cleavage reaction has been demonstrated using 40-base-pair and 7164-base-pair DNA substrates. The DNA cleavage reaction is not inhibited by dam methylation of the DNA substrate. Such semisynthetic site-specific DNA cleavage agents have potential applications in chromosome mapping, cloning, and sequencing.