SEQUENCE-SPECIFICITY OF HOLLIDAY JUNCTION RESOLUTION - IDENTIFICATIONOF RUVC MUTANTS DEFECTIVE IN METAL-BINDING AND TARGET SITE RECOGNITION

The RuvC protein of Escherichia coli resolves Holliday intermediates i n recombination and DNA repair by a dual strand incision mechanism tar geted to specific DNA sequences located symmetrically at the crossover . Two classes of amino acid substitutions are described that provide n ew insights into the sequence-specificity of the resolution reaction. The first includes D7N and G14S, which modify or eliminate metal bindi ng and prevent catalysis. The second, defined by G114D, G114N, and A11 6T, interfere with the ability of RuvC to cleave at preferred sequence s, but allow resolution at non-consensus target sites. All five mutant proteins bind junction DNA and impose an open conformation. D7N and:G 14S fail to induce hypersensitivity to hydroxyl radicals, a property o f RuvC previously thought to reflect junction opening. A different mec hanism is proposed whereby ferrous ions are co-ordinated in the comple x to induce a high local concentration of radicals. The open structure imposed by wild-type RuvC in Mg2+ is similar to that observed previou sly using a junction with a different stacking preference. G114D and A 116T impose slightly altered structures. This subtle change may be suf ficient to explain the failure of these proteins to cleave the sequenc es normally preferred. Gly114 and Ala116 residues Link two a-helices l ining the wall of the catalytic cleft in each subunit of RuvC. We sugg est that substitutions at these positions realign these helices and in terfere with the ability to establish base-specific contacts at resolu tion hotspots. (C) 1998 Academic Press