Functional interactions of Mycobacterium leprae RuvA with Escherichia coli RuvB and RuvC on Holliday junctions
Functional interactions of Mycobacterium leprae RuvA with Escherichia coli RuvB and RuvC on Holliday junctions
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DOI：
10.1006/jmbi.2000.4009
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发表时间：
2000-08-25
影响因子：
5.6
通讯作者：
Tsaneva, IR
中科院分区：
文献类型：
--
作者：
Arenas-Licea, J;van Gool, AJ;Tsaneva, IR
将麻风分枝杆菌RuvA同源物（MlRuvA）在大肠杆菌中过表达并纯化至均匀性。利用合成Holliday连接检测MlRuvA与大肠杆菌RuvB和RuvC （EcRuvB和EcRuvC）的dna结合特异性和功能相互作用。MlRuvA与Holliday节点结合，产生与EcRuvA相似的带移模式。此外，MlRuvA与EcRuvB形成了功能性的DNA解旋酶和分支迁移酶，尽管外源酶的效率较低。这些结果表明麻风分枝杆菌RuvA同源物是一个功能性分支迁移亚基。尽管MlRuvA与EcRuvB结合促进了分支迁移，但它无法在RuvABC复合体中刺激分支迁移依赖的解决方案。不能刺激RuvC并不是因为它不能在连接上形成异源RuvABC复合物，因为这种复合物是通过共免疫沉淀检测到的。最有可能的是，异种RuvABC复合物的稳定性以及RuvA和RuvC之间的相互作用受到损害，因为凝胶移位实验未能显示混合的mlruva - ecruvc连接复合物。这些结果表明，分支迁移本身和RuvABC复合物在Holliday连接上的组装不足以使RuvABC依赖ruvab的连接被RuvC分解，这表明RuvABC“分解体”的功能需要所有三种蛋白质之间特异性和密切的相互作用。(C) 2000年学术出版社。
The Mycobacterium leprae RuvA homologue (MlRuvA) was over-expressed in Escherichia coli and purified to homogeneity. The DNA-binding specificity and the functional interactions of MlRuvA with E. coli RuvB and RuvC (EcRuvB and EcRuvC) were examined using synthetic Holliday junctions. MlRuvA bound specifically to Holliday junctions and produced similar band-shift patterns as EcRuvA. Moreover, MlRuvA formed functional DNA helicase and branch-migration enzymes with EcRuvB, although the heterologous enzyme had a lower efficiency. These results demonstrate that the RuvA homologue of M. leprae is a functional branch-migration subunit.Whereas MlRuvA promoted branch-migration in combination with EcRuvB, it was unable to stimulate branch-migration-dependent resolution in a RuvABC complex. The inability to stimulate RuvC was not due to its failure to form heterologous RuvABC complexes on junctions, since such complexes were detected by co-immunoprecipitation. Most likely, the stability of the heterologous RuvABC complex and, possibly, the interactions between RuvA and RuvC were impaired, as gel-shift experiments failed to show mixed MlRuvA-EcRuvC-junction complexes. These results demonstrate that branch-migration per se and the assembly of a RuvABC complex on the Holliday junction are insufficient for RuvAB-dependent resolution of the junction by RuvC, suggesting that specific and intimate interactions between all three proteins are required for the function of a RuvABC "resolvasome". (C) 2000 Academic Press.