Abstract
The RusA protein of Escherichia coli is an endonuclease that resolves Holliday intermediates in recombination and DNA repair. Analysis of its subunit structure revealed that the native protein is a dimer. Its resolution activity was investigated using synthetic X-junctions with homologous cores. Resolution occurs by dual strand incision predominantly 5' of CC dinucleotides located symmetrically. A junction lacking homology is not resolved. The efficiency of resolution is related inversely to the number of base pairs in the homologous core, which suggests that branch migration is rate-limiting. Inhibition of resolution at high ratios of protein to DNA suggests that binding of RusA may immobilize the junction point at non-cleavable sites. Resolution is stimulated by alkaline pH and by Mn2+. The protein is unstable in the absence of substrate DNA and loses approximately 80% of its activity within 1 min under standard reaction conditions. DNA binding stabilizes the activity. Junction resolution is inhibited in the presence of RuvA. This observation probably explains why RusA is unable to promote efficient recombination and DNA repair in ruvA+ strains unless it is expressed at a high level.
Highlights
The RusA protein of Escherichia coli is an endonuclease that resolves Holliday intermediates made during homologous genetic recombination and DNA repair [1]
Purification and Physical Analysis of RusA—In previous work, we described a recombinant plasmid for the overexpression of RusA and purified some of the protein from strain N3757 transformed with this construct [1]
Effect of Homologous Core Size—The results described in the previous sections revealed that factors other than the presence of a target sequence can have an overriding effect on the efficiency of resolution by RusA
Summary
The RusA protein of Escherichia coli is an endonuclease that resolves Holliday intermediates made during homologous genetic recombination and DNA repair [1]. RusA was discovered through its ability to promote recombination and DNA repair in strains lacking RuvC Genetic analysis of this alternative resolvase provided the first indication that RuvC cannot cleave junctions efficiently in vivo without the associated activities of RuvA and RuvB [1, 30]. These two proteins assemble at junctions to form a highly specialized RuvAB complex that drives the branch point along the DNA [31,32,33,34]. We report its general biochemical properties, its sequence specificity, and the inhibition of its resolution activity by RuvA
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