Saha S, Rao D N
Department of Biochemistry, Indian Institute of Science, Bangalore.
J Mol Biol. 1997 Jun 13;269(3):342-54. doi: 10.1006/jmbi.1997.1045.
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.
III型限制修饰酶的Res亚基与所谓DEAD家族的几种RNA和DNA解旋酶在氨基酸序列上具有统计学上显著的相似性。据推测，在III型限制酶中，DNA解旋酶活性可能是在切割位点进行局部解旋所必需的。该家族成员共有七个保守基序，所有这些基序都存在于III型限制酶的Res亚基中。为了确定这些基序对III型限制酶EcoPI切割DNA的贡献（如果有的话），我们对基序I和II进行了改变。虽然基序I（GTGKT）中的突变明显影响ATP水解并导致DNA切割活性丧失，但基序II（DEPH）中的突变显著降低了ATP水解，但对DNA切割没有影响。双突变体R.EcoPIK90R-H229K尽管ATP结合未受影响，但未显示出明显的ATP酶或DNA限制活性。这些结果表明，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。我们认为这些基序在III型限制酶中被认为发生的ATP依赖性转位中起作用。
