Sequence-specificity of Holliday junction resolution: identification of RuvC mutants defective in metal binding and target site recognition.
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.
Hagan
N F
NF
Queens Medical Centre, University of Nottingham, Nottingham, NG7 2UH, UK.
Vincent
S D
SD
Ingleston
S M
SM
Sharples
G J
GJ
Bennett
R J
RJ
West
S C
SC
Lloyd
R G
RG
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D004260:DNA Repair; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins; D017665:Hydroxyl Radical / Q000737:chemistry; D046911:Macromolecular Substances; D008670:Metals / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D011995:Recombination, Genetic
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; D008670:Metals; C069806:ruvC protein, E coli; D017665:Hydroxyl Radical; D004706:Endodeoxyribonucleases