Target recognition by EcoKI:: The recognition domain is robust and restriction-deficiency commonly results from the proteolytic control of enzyme activity
Target recognition by EcoKI:: The recognition domain is robust and restriction-deficiency commonly results from the proteolytic control of enzyme activity
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DOI：
10.1006/jmbi.2001.4543
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发表时间：
2001-03-30
影响因子：
5.6
通讯作者：
Murray, NE
中科院分区：
文献类型：
--
作者：
O'Neill, M;Powell, LM;Murray, NE
我们报道了一种I型限制性内切和修饰酶EcoKI的靶识别结构域（TRD）的遗传和生化分析。I型R-M系统的trd位于特异性亚基（HsdS）内，HsdS赋予具有限制性和修饰活性的复合物序列特异性。随机突变表明，在EcoKI的一个由157个氨基酸残基组成的区域的氨基TRD中，大多数替换对细菌的表型没有可检测到的影响，即使这些替换是非保守的。TRD的结构似乎很坚固。在6个具有限制性缺陷、修饰缺陷(r（-)m(-)）表型的替换中，除了一个之外，其余的替换都位于残基80和110之间的区间，该区域通过序列比较预测形成了蛋白质- dna界面的一部分。影响该间隔的其他位点定向突变通常会损害限制和修饰。然而，我们发现r（-）表型不能作为EcoKI复合物缺乏内切酶活性的证据；即使修饰效率轻微受损，EcoKI的内切酶活性也会被依赖于ClpXP蛋白酶的过程破坏。来自r(-)m（-）表型突变体的酶通常保留一些序列特异性活性；甲基化酶活性可以在半甲基化的DNA底物上检测到，当r(-)m（-）细菌的生存能力依赖于ClpXP时，就暗示了残留的内切酶活性。相反，ClpX(-) r(-)m（-）细菌的活力可以作为很少或没有内切酶活性的证据。在14个具有r(-)m（-）表型的突变体中，只有6个在没有ClpXP的情况下存活。六个残基中的四个（G91, G105， F107和G141）的重要性通过发现即使对这些残基进行保守替换也会损害修饰，从而赋予r(-)m（-）表型而得到增强。(C) 2001学术出版社。
We report a genetic and biochemical analysis of a target recognition domain (TRD) of EcoKI, a type I restriction and modification enzyme. The TRDs of type I R-M systems are within the specificity subunit (HsdS) and HsdS confers sequence specificity to a complex endowed with both restriction and modification activities. Random mutagenesis has revealed that most substitutions within the amino TRD of EcoKI, a region comprising 157 amino acid residues, have no detectable effect on the phenotype of the bacterium, even when the substitutions are nonconservative. The structure of the TRD appears to be robust. All but one of the six substitutions that confer a restriction-deficient, modification-deficient (r(-)m(-)) phenotype were found to be in the interval between residues 80 and 110, a region predicted by sequence comparisons to form part of the protein-DNA interface. Additional site-directed mutations affecting this interval commonly impair both restriction and modification. However, we show that an r(-) phenotype cannot be taken as evidence that the EcoKI complex lacks endonuclease activity; in response to even a slightly impaired modification efficiency, the endonuclease activity of EcoKI is destroyed by a process dependent upon the ClpXP protease.Enzymes from mutants with an r(-)m(-) phenotype commonly retain some sequence-specific activity; methylase activity can be detected on hemimethylated DNA substrates and residual endonuclease activity is implied whenever the viability of the r(-)m(-) bacterium is dependent on ClpXP. Conversely, the viability of ClpX(-) r(-)m(-) bacteria can be used as evidence for little, or no, endonuclease activity. Of 14 mutants with an r(-)m(-) phenotype, only six are viable in the absence of ClpXP. The significance of four of the six residues (G91, G105, F107 and G141) is enhanced by the finding that even conservative substitutions for these residues impair modification, thereby conferring an r(-)m(-) phenotype. (C) 2001 Academic Press.