Arenas-Licea, J; van Gool, AJ; Keeley, AJ; Davies, A; West, SC; Tsaneva, IR; (2000) Functional interactions of Mycobacterium leprae RuvA with Escherichia coli RuvB and RuvC on Holliday junctions. J MOL BIOL , 301 (4) 839 - 850. 10.1006/jmbi.2000.4009.
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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.
|Title:||Functional interactions of Mycobacterium leprae RuvA with Escherichia coli RuvB and RuvC on Holliday junctions|
|Keywords:||recombination, Holliday junctions, branch-migration, RuvABC, DNA HELICASE ACTIVITY, BRANCH MIGRATION, IN-VITRO, SCHIZOSACCHAROMYCES-POMBE, SUBSTRATE-SPECIFICITY, GENETIC-RECOMBINATION, CRYSTAL-STRUCTURE, PROTEIN RUVA, RESOLUTION, COMPLEX|
|UCL classification:||UCL > School of Life and Medical Sciences > Faculty of Life Sciences|
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