一种用于归巢内切核酸酶活性的体内筛选系统。
An in vivo selection system for homing endonuclease activity.
作者信息
Gruen Mathias, Chang Kathy, Serbanescu Irina, Liu David R
机构信息
Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, MA 02138, USA.
出版信息
Nucleic Acids Res. 2002 Apr 1;30(7):e29. doi: 10.1093/nar/30.7.e29.
Abstract
Homing endonucleases are enzymes that catalyze the highly sequence-specific cleavage of DNA. We have developed an in vivo selection in Escherichia coli that links cell survival with homing endonuclease-mediated DNA cleavage activity and sequence specificity. Using this selection, wild-type and mutant variants of three homing endonucleases were characterized without requiring protein purification and in vitro analysis. This selection system may facilitate the study of sequence-specific DNA cleaving enzymes, and selections based on this work may enable the evolution of homing endonucleases with novel activities or specificities.
摘要
归巢内切酶是催化DNA进行高度序列特异性切割的酶。我们开发了一种在大肠杆菌中的体内筛选方法，该方法将细胞存活与归巢内切酶介导的DNA切割活性及序列特异性联系起来。利用这种筛选方法，无需蛋白质纯化和体外分析就对三种归巢内切酶的野生型和突变体变体进行了表征。这种筛选系统可能有助于对序列特异性DNA切割酶的研究，基于这项工作的筛选可能会使具有新活性或特异性的归巢内切酶得以进化。
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The monomeric homing endonuclease PI-SceI has two catalytic centres for cleavage of the two strands of its DNA substrate.单体归巢内切酶PI-SceI有两个催化中心，用于切割其DNA底物的两条链。
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