SEQUENCE SPECIFICITY AND BIOCHEMICAL-CHARACTERIZATION OF THE RUSA HOLLIDAY JUNCTION RESOLVASE OF ESCHERICHIA-COLI

The RusA protein of Escherichia coli is an endonuclease that resolves Holliday intermediates in recombination and DNA repair. Analysis of it s subunit structure revealed that the native protein is a dimer. Its r esolution activity was investigated using synthetic X-junctions with h omologous cores. Resolution occurs by dual strand incision predominant ly 5' of CC dinucleotides located symmetrically. A junction lacking ho mology is not resolved. The efficiency of resolution is related invers ely to the number of base pairs in the homologous core, which suggests that branch migration is rate-limiting. Inhibition of resolution at h igh ratios of protein to DNA suggests that binding of RusA may immobil ize the junction point at non-cleavable sites. Resolution is stimulate d by alkaline pH and by Mn2+. The protein is unstable in the absence o f substrate DNA and loses similar to 80% of its activity within 1 min under standard reaction conditions. DNA binding stabilizes the activit y. Junction resolution is inhibited in the presence of RuvA. This obse rvation probably explains why RusA is unable to promote efficient reco mbination and DNA repair in ruvA(+) strains unless it is expressed at a high level.