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首页> 外文期刊>Journal of Molecular Biology >MUTATIONS IN THE RES SUBUNIT OF THE ECOPI RESTRICTION ENZYME THAT AFFECT ATP-DEPENDENT REACTIONS
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MUTATIONS IN THE RES SUBUNIT OF THE ECOPI RESTRICTION ENZYME THAT AFFECT ATP-DEPENDENT REACTIONS

机译：影响ATP依赖反应的ECOPI限制酶的RES亚基的突变

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The Res subunits of the type III restriction-modification enzymes share a statistically significant amino acid sequence similarity with several RNA and DNA helicases of the so-called DEAD family. It was postulated that in type III restriction enzymes a DNA helicase activity may be required for local unwinding at the cleavage site. The members of this family share seven conserved motifs, all of which are found in the Res subunit of the type III restriction enzymes. To determine the contribution, if any, of these motifs in DNA cleavage by EcoPI, a type III restriction enzyme, we have made changes in motifs I and II. While mutations in motif I (GTGKT) clearly affected ATP hydrolysis and resulted in loss of DNA cleavage activity, mutation in motif II (DEPH) significantly decreased ATP hydrolysis but had no effect on DNA cleavage. The double mutant R.EcoPIK90R-H229K showed no significant ATPase or DNA restriction activity though ATP binding was not affected. These results imply that there are at least two ATPase reaction centres in EcoPI restriction enzyme. Motif I appears to be involved in coupling DNA restriction to ATP hydrolysis. Our results indicate that EcoPI restriction enzyme does not have a strand separation activity. We suggest that these motifs play a role in the ATP-dependent translocation that has been proposed to occur in the type III restriction enzymes. (C) 1997 Academic Press Limited. [References: 35]
机译：III型限制性修饰酶的Res亚基与所谓的DEAD家族的几种RNA和DNA解旋酶具有统计学上显着的氨基酸序列相似性。据推测，在III型限制酶中，可能需要DNA解旋酶活性以在切割位点局部解开。该家族的成员共有七个保守的基序，所有这些基序都存在于III型限制酶的Res亚基中。为了确定这些基序在通过III型限制性酶EcoPI切割DNA中的贡献，如果有的话，我们对基序I和II进行了更改。尽管基序I的突变（GTGKT）明显影响ATP水解并导致DNA裂解活性的丧失，但基序II的突变（DEPH）显着降低了ATP水解，但对DNA裂解没有影响。尽管不影响ATP结合，但双突变体R.EcoPIK90R-H229K没有明显的ATPase或DNA限制性酶活性。这些结果暗示在EcoPI限制酶中至少有两个ATP酶反应中心。基序I似乎参与将DNA限制酶与ATP水解偶联。我们的结果表明，EcoPI限制酶不具有链分离活性。我们建议这些基序在已提出要在III型限制酶中发生的ATP依赖性易位中起作用。 （C）1997 Academic Press Limited。 [参考：35]

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