Kim Y G, Smith J, Durgesha M, Chandrasegaran S
Department of Environmental Health Sciences, The Johns Hopkins University, School of Medicine, Baltimore, Maryland 21205-2179, USA.
Biol Chem. 1998 Apr-May;379(4-5):489-95. doi: 10.1515/bchm.1998.379.4-5.489.
Gal4, a yeast protein, activates transcription of genes required for metabolism of galactose and melibiose. It binds as a dimer to a consensus palindromic 17-base pair DNA sequence. It is a member of the third family of proteins that contain zinc-mediated peptide loops that interact specifically with nucleic acids. Gal4 has a very distinctive zinc coordination profile and mode of DNA-binding. Here, we report the creation of a novel site-specific endonuclease by linking the N-terminal 147 amino acids of Gal4 to the cleavage domain of FokI endonuclease. The fusion protein is active and under optimal conditions, binds to a 17 bp consensus DNA site and cleaves near this site. As expected, the cleavage occurs on either side of the consensus binding site(s).
Gal4是一种酵母蛋白，可激活半乳糖和蜜二糖代谢所需基因的转录。它以二聚体形式结合到一个共有回文17碱基对的DNA序列上。它是第三类蛋白质家族的成员，这类蛋白质含有锌介导的肽环，可与核酸特异性相互作用。Gal4具有非常独特的锌配位特征和DNA结合模式。在此，我们报告通过将Gal4的N端147个氨基酸与FokI核酸内切酶的切割结构域相连，创建了一种新型位点特异性核酸内切酶。融合蛋白具有活性，在最佳条件下，可结合到一个17bp的共有DNA位点并在该位点附近切割。正如预期的那样，切割发生在共有结合位点的两侧。
