Functional interactions of Mycobacterium leprae RuvA with Escherichia coli RuvB and RuvC on holliday junctions.
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".
Arenas-Licea
J
J
Department of Biochemistry and Molecular Biology, University College London, London, WC1E 6BT, UK.
van Gool
A J
AJ
Keeley
A J
AJ
Davies
A
A
West
S C
SC
Tsaneva
I R
IR
D000251:Adenosine Triphosphatases / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002413:Cations, Divalent / Q000378:metabolism / Q000494:pharmacology; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004265:DNA Helicases / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004706:Endodeoxyribonucleases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; D008274:Magnesium / Q000378:metabolism / Q000494:pharmacology; D009166:Mycobacterium leprae / Q000201:enzymology* / Q000235:genetics; D011485:Protein Binding / Q000187:drug effects; D011995:Recombination, Genetic / Q000235:genetics; D013379:Substrate Specificity
D001426:Bacterial Proteins; D002413:Cations, Divalent; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; C061260:RuvB protein, Bacteria; C069806:ruvC protein, E coli; D004247:DNA; D004706:Endodeoxyribonucleases; C071059:Holliday junction DNA helicase, E coli; D000251:Adenosine Triphosphatases; D004265:DNA Helicases; D008274:Magnesium; D002118:Calcium