Macmaster Rachel, Sedelnikova Svetlana, Baker Patrick J, Bolt Edward L, Lloyd Robert G, Rafferty John B
Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, UK.
Nucleic Acids Res. 2006;34(19):5577-84. doi: 10.1093/nar/gkl447. Epub 2006 Oct 5.
We have determined the structure of a catalytically inactive D70N variant of the Escherichia coli RusA resolvase bound to a duplex DNA substrate that reveals critical protein-DNA interactions and permits a much clearer understanding of the interaction of the enzyme with a Holliday junction (HJ). The RusA enzyme cleaves HJs, the fourway DNA branchpoints formed by homologous recombination, by introducing symmetrical cuts in the phosphodiester backbone in a Mg2+ dependent reaction. Although, RusA shows a high level of selectivity for DNA junctions, preferring to bind fourway junctions over other substrates in vitro, it has also been shown to have appreciable affinity for duplex DNA. However, RusA does not show DNA cleavage activity with duplex substrates. Our structure suggests the possible basis for structural selectivity as well as sources of the sequence specificity observed for DNA cleavage by RusA.
我们已经确定了与双链DNA底物结合的大肠杆菌RusA解离酶催化失活的D70N变体的结构，该结构揭示了关键的蛋白质-DNA相互作用，并能更清楚地理解该酶与霍利迪连接体（HJ）的相互作用。RusA酶通过在Mg2+依赖性反应中在磷酸二酯主链上引入对称切割来切割HJ，即由同源重组形成的四链DNA分支点。虽然RusA对DNA连接体表现出高度的选择性，在体外更倾向于结合四链连接体而非其他底物，但也已证明它对双链DNA具有相当的亲和力。然而，RusA对双链底物不表现出DNA切割活性。我们的结构表明了结构选择性的可能基础以及RusA切割DNA时观察到的序列特异性的来源。
