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首页> 外文期刊>Biological chemistry >Isolation of a Non-Classical Mutant of the DNA Recognition Subunit of the Type I Restriction Endonuclease R.EcoR124I
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Isolation of a Non-Classical Mutant of the DNA Recognition Subunit of the Type I Restriction Endonuclease R.EcoR124I

机译：I型限制性内切核酸酶R.EcoR124I的DNA识别亚基的非经典突变体的分离

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摘要

We have used deletion mutagenesis and PCR-bases misincorporation mutagenesis to produce a collection of mutations in the central conserved region of the DNA binding subunit of the type IC restriction endonuclease EcoR124I. It has been proposed that this domain is involved in protein-protein interactions during the assembly of the endonuclease. While a large percentage of these mutations gave a classical Res~- Mod~- phenotype, one mutant was isolated with a nonclassical Res~- Mod~+ phenotype. The loss of restriction activity, but retention of the ability to modify indicates that this mutation cannot affect DNA binding and must alter the assembly of the endonuclease in such a way as to prevent DNA cleavage but allow methylation. This mutant resulted from a single amino acid change Trp~(212) → Arg. The location of the single amino acid change is at the border of the central conserved region and the second target recognition domain (TRD2) and suggests that this region is extremely important for the assembly of the methylase with the HsdR subunit into a functional restriction endonuclease.
机译：我们已使用缺失诱变和PCR碱基错误掺入诱变在IC型限制性核酸内切酶EcoR124I的DNA结合亚基的中央保守区中产生了一系列突变。已经提出，该结构域在核酸内切酶组装过程中参与蛋白质-蛋白质相互作用。尽管这些突变中有很大一部分具有经典的Res--Mod--表型，但分离出一个具有非经典Res--Mod- +表型的突变体。限制性酶活性的丧失，但保留了修饰的能力，表明该突变不能影响DNA的结合，必须以防止DNA裂解但允许甲基化的方式改变核酸内切酶的组装。该突变体是由单个氨基酸变化Trp_（212）→Arg引起的。单个氨基酸变化的位置位于中央保守区和第二个靶标识别域（TRD2）的边界，这表明该区域对于将具有HsdR亚基的甲基化酶组装成功能性限制性核酸内切酶极为重要。

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