Xu S Y, Schildkraut I
New England Biolabs, Beverly, MA 01915.
Biotechniques. 1993 Aug;15(2):310-5.
This report describes the use of a biochemical tool that has been developed to aid in the manipulation of DNA. A DNA binding-proficient and cleavage-deficient BamHI mutant protein, E113K, was used in vitro to protect its recognition sequence (5'-GGATCC-3') against the catalytic action of site-specific endonuclease, exonuclease and methylase. In vitro conditions are reported here in which the E113K protein protects BamHI sites (5'-GGATCC-3') from cleavage by BamHI endonuclease or Sau3AI endonuclease (5'-GATC-3'); protects a neighboring restriction site 5'-CCCGGG-3' from SmaI endonuclease digestion; blocks methylation of 5'-GGATCC-3' by Dam methylase (5'-GATC-3'); and blocks Bal31 exonuclease progression at a BamHI site. The Bal31 procedure could be used to generate unidirectional deletions of a DNA fragment. The use of mutant endonucleases that are binding-proficient and cleavage-deficient to shield DNA from nuclease digestion or methylase modification expands the repertoire of methods to manipulate DNA in vitro.
本报告描述了一种已开发出来用于辅助DNA操作的生化工具的应用。一种具有DNA结合能力但缺乏切割活性的BamHI突变蛋白E113K，在体外被用于保护其识别序列（5'-GGATCC-3'）免受位点特异性内切酶、核酸外切酶和甲基化酶的催化作用。本文报道了体外条件下，E113K蛋白保护BamHI位点（5'-GGATCC-3'）不被BamHI内切酶或Sau3AI内切酶（5'-GATC-3'）切割；保护相邻的限制酶切位点5'-CCCGGG-3'不被SmaI内切酶消化；阻止Dam甲基化酶（5'-GATC-3'）对5'-GGATCC-3'的甲基化；并阻止Bal31核酸外切酶在BamHI位点的作用进程。Bal31方法可用于产生DNA片段的单向缺失。使用具有结合能力但缺乏切割活性的突变内切酶来保护DNA免受核酸酶消化或甲基化酶修饰，扩展了体外操作DNA的方法种类。
