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Sequence-specificity of Holliday junction resolution: identification of RuvC mutants defective in metal binding and target site recognition.

Hagan, N.F.; Vincent, S.D.; Ingleston, S.M.; Sharples, G.J.; Bennett, R.J.; West, S.C.; Lloyd, R.G.

Authors

N.F. Hagan

S.D. Vincent

S.M. Ingleston

R.J. Bennett

S.C. West

R.G. Lloyd



Abstract

The RuvC protein of Escherichia coli resolves Holliday intermediates in recombination and DNA repair by a dual strand incision mechanism targeted to specific DNA sequences located symmetrically at the crossover. Two classes of amino acid substitutions are described that provide new insights into the sequence-specificity of the resolution reaction. The first includes D7N and G14S, which modify or eliminate metal binding and prevent catalysis. The second, defined by G114D, G114N, and A116T, interfere with the ability of RuvC to cleave at preferred sequences, but allow resolution at non-consensus target sites. All five mutant proteins bind junction DNA and impose an open conformation. D7N and G14S fail to induce hypersensitivity to hydroxyl radicals, a property of RuvC previously thought to reflect junction opening. A different mechanism is proposed whereby ferrous ions are co-ordinated in the complex to induce a high local concentration of radicals. The open structure imposed by wild-type RuvC in Mg2+ is similar to that observed previously using a junction with a different stacking preference. G114D and A116T impose slightly altered structures. This subtle change may be sufficient to explain the failure of these proteins to cleave the sequences normally preferred. Gly114 and Ala116 residues link two alpha-helices lining the wall of the catalytic cleft in each subunit of RuvC. We suggest that substitutions at these positions realign these helices and interfere with the ability to establish base-specific contacts at resolution hotspots.

Citation

Hagan, N., Vincent, S., Ingleston, S., Sharples, G., Bennett, R., West, S., & Lloyd, R. (1998). Sequence-specificity of Holliday junction resolution: identification of RuvC mutants defective in metal binding and target site recognition. Journal of Molecular Biology, 281, 17-29

Journal Article Type Article
Publication Date 1998
Journal Journal of Molecular Biology
Print ISSN 0022-2836
Publisher Elsevier
Volume 281
Pages 17-29
Publisher URL http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=9680472