Differential binding of wild-type and a mutant RepA protein to oriR sequence suggests a model for the initiation of plasmid R1 replication.
DNA replication of the enterobacterial plasmid R1 is initiated by RepA protein. We have developed a new procedure for the purification of RepA from inclusion bodies, which involves CHAPS-mediated solubilization. This method has been also used for the thermosensitive mutant protein RepA2623. The nucleoprotein complexes obtained with both proteins and oriR, the origin of replication, are studied in this paper. DNaseI and hydroxyl-radical footprinting suggest the presence in oriR of two sites with different affinity for RepA separated by eight helical turns. The pattern of hypersensitive sites in the footprints indicates that the oriR sequence, when complexed with RepA, is curved. The binding of RepA molecules to oriR is co-operative and this co-operativity is defective in the thermosensitive protein. Band-shift analysis of RepA-oriR complexes revealed the existence of a species with an anomalously high electrophoretic mobility that appears after formation of the first RepA-oriR complex and requires the sequential interaction of RepA with its two distal binding sites. These features lead us to propose that protein-protein interactions between RepA bound to both distal sites could be responsible for oriR looping. This model represents a novel mechanism that results in activation of an origin in a replicon that does not contain iterons.
Giraldo
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R
Unidad de Ingeniería Genética, Centro de Investigaciones Biológicas-CSIC, Madrid, Spain.
Díaz
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D001426:Bacterial Proteins / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D004265:DNA Helicases; D004261:DNA Replication; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins; D003850:Deoxyribonuclease I / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics*; D005609:Free Radicals; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D006878:Hydroxides; D046911:Macromolecular Substances; D008958:Models, Molecular; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D009698:Nucleoproteins / Q000187:drug effects / Q000378:metabolism; D010957:Plasmids / Q000235:genetics / Q000378:metabolism*; D011489:Protein Denaturation / Q000187:drug effects; D011506:Proteins; D012689:Sequence Homology, Nucleic Acid; D012995:Solubility; D015534:Trans-Activators
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D005609:Free Radicals; D006146:Guanidines; D006878:Hydroxides; D046911:Macromolecular Substances; D009698:Nucleoproteins; D011506:Proteins; D015534:Trans-Activators; C045264:replication initiator protein; D003850:Deoxyribonuclease I; D004265:DNA Helicases; D019791:Guanidine