Activating mutations of Tn3 resolvase marking interfaces important in recombination catalysis and its regulation
Activating mutations of Tn3 resolvase marking interfaces important in recombination catalysis and its regulation
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DOI：
10.1046/j.1365-2958.2003.03831.x
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发表时间：
2004-02-01
影响因子：
3.6
通讯作者：
Stark, WM
中科院分区：
文献类型：
--
作者：
Burke, ME;Arnold, PH;Stark, WM
Tn 3解离酶对DNA重组的催化作用取决于突触的预先形成，该突触包含12个解离酶亚基和两个重组位点（res）。每个res在发生链交换的位点I结合一个解离酶二聚体，在两个相邻的“辅助”结合位点II和III结合另外的二聚体。在E.杆菌一些单突变体可以解析res x site I质粒（即，具有一个res和一个site I），但两个或更多个激活突变对于有效解析site I x site I质粒是必需的。通过过度活跃的突变体的位点I X位点I解析可以通过在晶体学2-3'界面处的突变进一步刺激，所述突变消除野生型解离酶的活性。激活突变可以允许野生型系统的调节机制被绕过，通过稳定或去稳定突触中亚基内和亚基之间的界面。突变的位置和特征支持丝氨酸重组酶的链交换机制，其中DNA在重组酶四聚体的外部，并且四聚体的三级/四级结构被重新配置。
Catalysis of DNA recombination by Tn3 resolvase is conditional on prior formation of a synapse, comprising 12 resolvase subunits and two recombination sites (res). Each res binds a resolvase dimer at site I, where strand exchange takes place, and additional dimers at two adjacent 'accessory' binding sites II and III. 'Hyperactive' resolvase mutants, that catalyse strand exchange at site I without accessory sites, were selected in E. coli. Some single mutants can resolve a res x site I plasmid (that is, with one res and one site I), but two or more activating mutations are necessary for efficient resolution of a site I x site I plasmid. Site I x site I resolution by hyperactive mutants can be further stimulated by mutations at the crystallographic 2-3' interface that abolish activity of wild-type resolvase. Activating mutations may allow regulatory mechanisms of the wild-type system to be bypassed, by stabilizing or destabilizing interfaces within and between subunits in the synapse. The positions and characteristics of the mutations support a mechanism for strand exchange by serine recombinases in which the DNA is on the outside of a recombinase tetramer, and the tertiary/quaternary structure of the tetramer is reconfigured.