de la Tour Claire Bouthier, Amrani Laila, Cossard Raynald, Neuman Keir C, Serre Marie Claude, Duguet Michel
Université Paris-Sud 11, UMR8621 Institut de Génétique et Microbiologie, 91405 Orsay Cedex, France.
Université Paris-Sud 11, UMR8621 Institut de Génétique et Microbiologie, 91405 Orsay Cedex, France.
J Biol Chem. 2008 Oct 10;283(41):27395-27402. doi: 10.1074/jbc.M800867200. Epub 2008 Jul 8.
Reverse gyrase is a unique type IA topoisomerase that is able to introduce positive supercoils into DNA in an ATP-dependent process. ATP is bound to the helicase-like domain of the enzyme that contains most of the conserved motifs found in helicases of the SF1 and SF2 superfamilies. In this paper, we have investigated the role of the conserved helicase motifs I, II, V, VI, and Q by generating mutants of the Thermotoga maritima reverse gyrase. We show that mutations in motifs I, II, V, and VI completely eliminate the supercoiling activity of reverse gyrase and that a mutation in the Q motif significantly reduces this activity. Further analysis revealed that for most mutants, the DNA binding and cleavage properties are not significantly changed compared with the wild type enzyme, whereas their ATPase activity is impaired. These results clearly show that the helicase motifs are tightly involved in the coupling of ATP hydrolysis to the topoisomerase activity. The zinc finger motif located at the N-terminal end of reverse gyrases was also mutated. Our results indicate that this motif plays an important role in DNA binding.
反向回旋酶是一种独特的IA型拓扑异构酶，能够在依赖ATP的过程中将正超螺旋引入DNA。ATP与该酶的解旋酶样结构域结合，该结构域包含在SF1和SF2超家族解旋酶中发现的大多数保守基序。在本文中，我们通过构建嗜热栖热菌反向回旋酶的突变体，研究了保守解旋酶基序I、II、V、VI和Q的作用。我们发现，基序I、II、V和VI中的突变完全消除了反向回旋酶的超螺旋活性，而Q基序中的突变显著降低了这种活性。进一步分析表明，对于大多数突变体，与野生型酶相比，其DNA结合和切割特性没有显著变化，但其ATP酶活性受损。这些结果清楚地表明，解旋酶基序紧密参与了ATP水解与拓扑异构酶活性的偶联。位于反向回旋酶N末端的锌指基序也发生了突变。我们的结果表明，该基序在DNA结合中起重要作用。
