Quantifying the information content of homing endonuclease target sites by single base pair profiling.
Quantifying the information content of homing endonuclease target sites by single base pair profiling.
复制标题
通过单碱基对分析量化归巢核酸内切酶靶位点的信息内容。
DOI：
10.1007/978-1-62703-968-0_11
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发表时间：
2014
期刊：
影响因子：
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通讯作者：
MonnatJr,RaymondJ
中科院分区：
文献类型：
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作者：
Friedman,JoshuaI;Li,Hui;MonnatJr,RaymondJ
归巢核酸内切酶（Homing endonuclease，HE）是一种在体内外都能识别长DNA序列的天然蛋白质，具有高度的位点特异性。HE的靶位点特异性高，但不是绝对的。例如，归巢核酸内切酶（LHE）的充分表征的LAGLIDADG家族的成员识别约20个碱基对的靶位点，并且可以耐受一些靶位点碱基对变化而不丧失位点结合或切割活性。这种适度程度的靶位点简并性一旦被定义就实际上是有用的，并且可以促进具有新的DNA识别特异性的LHE变体的工程化。在这一章中，我们概述了一般协议，系统地分析HE靶位点碱基对的位置，以确定其功能的重要性，在体外和体内，并显示如何信息理论可以用来使所产生的数据的意义。
Homing endonucleases (HEs) are native proteins that recognize long DNA sequences with high site specificity in vitro and in vivo. The target site specificity of HEs is high, though not absolute. For example, members of the well-characterized LAGLIDADG family of homing endonucleases (the LHEs) recognize target sites of ~20 base pairs, and can tolerate some target site base pair changes without losing site binding or cleavage activity. This modest degree of target site degeneracy is practically useful once defined and can facilitate the engineering of LHE variants with new DNA recognition specificities. In this chapter, we outline general protocols for systematically profiling HE target site base pair positions in order to define their functional importance in vitro and in vivo, and show how information theory can be used to make sense of the resulting data.