ABSTRACT
Holliday junctions are four-way branched DNA structures formed during recombination, replication and repair. They are processed in Escherichia coli by the RuvA, RuvB and RuvC proteins. RuvA targets the junction and facilitates loading of RuvB helicase and RuvC endonuclease to form complexes that catalyse junction branch migration (RuvAB) and resolution (RuvABC). We investigated the role of RuvA in these reactions and in particular the part played by the acidic pin located on its DNA-binding surface. By making appropriate substitutions of two key amino acids (Glu55 and Asp56), we altered the charge on the pin and investigated how this affected junction binding and processing. We show that two negative charges on each subunit of the pin are crucial. They facilitate junction targeting by preventing binding to duplex DNA and also constrain branch migration by RuvAB in a manner critical for junction processing. These findings provide the first direct evidence that RuvA has a mechanistic role in branch migration. They also provide insight into the coupling of branch migration and resolution by the RuvABC resolvasome.
Subject(s)
DNA Helicases , DNA, Bacterial/chemistry , DNA, Bacterial/metabolism , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Escherichia coli Proteins , Escherichia coli/metabolism , Amino Acid Sequence , Bacterial Proteins/metabolism , Base Sequence , Conserved Sequence , DNA, Bacterial/genetics , DNA-Binding Proteins/genetics , Electrochemistry , Endodeoxyribonucleases/metabolism , Escherichia coli/genetics , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Nucleic Acid Conformation , Protein Conformation , Sequence Homology, Amino AcidSubject(s)
Bacteria/genetics , Bacterial Proteins/metabolism , Biological Evolution , DNA Helicases/metabolism , DNA, Bacterial/genetics , DNA, Bacterial/metabolism , DNA-Binding Proteins/metabolism , Endodeoxyribonucleases/metabolism , Escherichia coli Proteins , Holliday Junction Resolvases , Amino Acid Sequence , Bacterial Proteins/chemistry , DNA, Bacterial/chemistry , DNA-Binding Proteins/chemistry , Endodeoxyribonucleases/chemistry , Evolution, Molecular , Models, Molecular , Molecular Sequence Data , Nucleic Acid Conformation , Phylogeny , Protein Conformation , Sequence AlignmentABSTRACT
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.
Subject(s)
Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Endodeoxyribonucleases/genetics , Endodeoxyribonucleases/metabolism , Escherichia coli Proteins , Mutation , Amino Acid Sequence , Amino Acid Substitution , Bacterial Proteins/chemistry , Base Sequence , Binding Sites/genetics , DNA Repair , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , DNA, Bacterial/metabolism , Endodeoxyribonucleases/chemistry , Escherichia coli/genetics , Escherichia coli/metabolism , Hydroxyl Radical/chemistry , Macromolecular Substances , Metals/metabolism , Models, Molecular , Molecular Sequence Data , Nucleic Acid Conformation , Protein Conformation , Recombination, GeneticABSTRACT
Comparison of the structure of Escherichia coli RuvA with other proteins in the Protein Data Bank gives insights into the probable modes of association of RuvA with the Holliday junction during homologous recombination. All three domains of the RuvA protein possess striking structural similarities to other DNA-binding proteins. Additionally, the second domain of RuvA contains two copies of the helix-hairpin-helix (HhH) structural motif, which has been implicated in non-sequence-specific DNA binding. The two copies of the motif are related by approximate 2-fold symmetry and may form a bidentate DNA-binding module. The results described provide support for the organization of the arms of the DNA in our RuvA/Holliday junction complex model and support the involvement of the HhH motifs in DNA binding.
