PURIFICATION AND PROPERTIES OF THE RUVA AND RUVB PROTEINS OF ESCHERICHIA-COLI.
MOL GEN GENET
1 - 10.
The RuvA and RuvB proteins of Escherichia coli play important roles in the post-replicational repair of damaged DNA, genetic recombination and cell division. In this paper, we describe the construction of over expression vectors for RuvA and RuvB and detail simple purification schemes for each protein. The purified 22 kDa RuvA polypeptide forms a tetrameric protein (M(r) ca. 100000) as observed by gel filtration. The tetramer is stabilised by strong disulphide bridges that resist denaturation during SDS-PAGE (in the absence of boiling and beta-mercaptoethanol). In contrast, purified RuvB polypeptides (37 kDa) weakly associate to form a dimeric protein (M(r) ca. 85000). At low protein concentrations, the RuvB dimer dissociates into monomers. The multimeric forms of each protein may be covalently linked by the bifunctional cross-linking reagent dimethyl suberimidate. Addition of purified RuvA and RuvB to a RecA-mediated recombination reaction was found to stimulate the rate of strand exchange leading to the rapid formation of heteroduplex DNA.
|Title:||PURIFICATION AND PROPERTIES OF THE RUVA AND RUVB PROTEINS OF ESCHERICHIA-COLI|
|Keywords:||RECOMBINATION, DNA REPAIR, BRANCH MIGRATION, HOLLIDAY JUNCTION, MUTAGENESIS, HOLLIDAY JUNCTIONS INVITRO, DNA-REPAIR, GENE-PRODUCT, CONJUGATIONAL RECOMBINATION, K-12 REVEALS, RESOLUTION, MUTANTS, IDENTIFICATION, ENDONUCLEASE, EXPRESSION|
|UCL classification:||UCL > School of Life and Medical Sciences > Faculty of Life Sciences > Biosciences (Division of) > Structural and Molecular Biology|
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