Olorunniji Femi J, He Jiuya, Wenwieser Sandra V C T, Boocock Martin R, Stark W Marshall
Faculty of Biomedical & Life Sciences, University of Glasgow, Glasgow, Scotland, UK.
Nucleic Acids Res. 2008 Dec;36(22):7181-91. doi: 10.1093/nar/gkn885. Epub 2008 Nov 10.
The serine recombinase Tn3 resolvase catalyses recombination between two 114 bp res sites, each of which contains binding sites for three resolvase dimers. We have analysed the in vitro properties of resolvase variants with 'activating' mutations, which can catalyse recombination at binding site I of res when the rest of res is absent. Site I x site I recombination promoted by these variants can be as fast as res x res recombination promoted by wild-type resolvase. Activated variants have reduced topological selectivity and no longer require the 2-3' interface between subunits that is essential for wild-type resolvase-mediated recombination. They also promote formation of a stable synapse comprising a resolvase tetramer and two copies of site I. Cleavage of the DNA strands by the activated mutants is slow relative to the rate of synapsis. Stable resolvase tetramers were not detected in the absence of DNA or bound to a single site I. Our results lead us to conclude that the synapse is assembled by sequential binding of resolvase monomers to site I followed by interaction of two site I-dimer complexes. We discuss the implications of our results for the mechanisms of synapsis and regulation in recombination by wild-type resolvase.
丝氨酸重组酶Tn3解离酶催化两个114 bp的res位点之间的重组，每个res位点都包含三个解离酶二聚体的结合位点。我们分析了具有“激活”突变的解离酶变体的体外特性，当res的其余部分不存在时，这些变体可以在res的结合位点I处催化重组。这些变体促进的位点I×位点I重组可以与野生型解离酶促进的res×res重组一样快。激活的变体具有降低的拓扑选择性，并且不再需要野生型解离酶介导的重组所必需的亚基之间的2-3'界面。它们还促进形成由解离酶四聚体和两个位点I拷贝组成的稳定突触。相对于突触形成速率，激活的突变体对DNA链的切割较慢。在没有DNA或与单个位点I结合的情况下未检测到稳定的解离酶四聚体。我们的结果使我们得出结论，突触是通过解离酶单体与位点I的顺序结合，然后是两个位点I-二聚体复合物的相互作用而组装的。我们讨论了我们的结果对野生型解离酶重组中的突触形成和调控机制的影响。
