I-PpoI and I-CreI homing site sequence degeneracy determined by random mutagenesis and sequential in vitro enrichment
I-PpoI and I-CreI homing site sequence degeneracy determined by random mutagenesis and sequential in vitro enrichment
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DOI：
10.1006/jmbi.1998.1886
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发表时间：
1998-07-17
影响因子：
5.6
通讯作者：
Monnat, RJ
中科院分区：
文献类型：
--
作者：
Argast, GM;Stephens, KM;Monnat, RJ
构建了含有真核归巢内切酶I-PpoI和I-CreI部分随机切割位点的质粒文库，通过连续的切割和凝胶分离循环，在大肠杆菌中培养和生长，选择性地恢复了I-PpoI或I-CreI可以切割的位点。分离出21个不同的i - poi敏感归巢位点，包括原生归巢位点。这些位点在15bp归巢位点的4个核苷酸位置上是相同的，在其余11个位置上具有有限的碱基替换模式，并且表现出对位于归巢位点序列顶部链两侧的嘌呤的偏好。21个不同的i - crei敏感归巢点，包括原生点，被分离出来。在i - crei敏感的归巢位点变体中，有10个核苷酸位置相同，需要添加SDS才能有效释放裂解产物。这10个位置中有4个是相同的，在不需要产品发布SDS的主站。在归巢位点序列的顶端链两侧有一个优先的嘧啶。24个I-CreI归巢位点核苷酸位置中有3个明显缺乏信息含量，即当被任何核苷酸占据时都允许裂解。这些结果表明，I-PpoI和I-CreI在它们的归巢位点序列上建立了大量的dna -蛋白质接触，这些接触的不同亚群可能足以维持高度的序列特异性归巢位点识别和切割。我们使用的序列富集协议应该对定义序列退化和其他归巢内切酶目标位点的信息内容有用。(C) 1998学术出版社。
Plasmid libraries containing partially randomized cleavage sites for the eukaryotic homing endonucleases I-PpoI and I-CreI were constructed, and sites that could be cleaved by I-PpoI or I-CreI were selectively recovered by successive cycles of cleavage and gel separation followed by religation and growth in Escherichia coli. Twenty-one different I-PpoI-sensitive homing sites, including the native homing site, were isolated. These sites were identical at four nucleotide positions within the 15 bp homing site, had a restricted pattern of base substitutions at the remaining 11 positions and displayed a preference for purines flanking the top strand of the homing site sequence. Twenty-one different I-CreI-sensitive homing sites, including the native site, were isolated. Ten nucleotide positions were identical in homing site variants that were I-CreI-sensitive and required the addition of SDS for efficient cleavage product release. Four of these ten positions were identical in homing sites that did not require SDS for product release. There was a preference for pyrimidines flanking the top strand of the homing site sequence. Three of the 24 I-CreI homing site nucleotide positions apparently lacked informational content, i.e. were permissive of cleavage when occupied by any nucleotide. These results suggest that I-PpoI and I-CreI make a large number of DNA-protein contacts across their homing site sequences, and that different subsets of these contacts may be sufficient to maintain a high degree of sequence-specific homing site recognition and cleavage. The sequential enrichment protocol we used should be useful for defining the sequence degeneracy and informational content of other homing endonuclease target sites. (C) 1998 Academic Press.