Binding modes of the initiator and inhibitor forms of the replication protein pi to the gamma ori iteron of plasmid R6K.
Discerning the interactions between initiator protein and the origin of replication should provide insights into the mechanism of DNA replication initiation. In the gamma origin of plasmid R6K, the Rep protein, pi, is distinctive in that it can bind the seven 22-bp iterons in two forms; pi monomers activate replication, whereas pi dimers act as inhibitors. In this work, we used wild type and variants of the pi protein with altered monomer/dimer ratios to study iteron/pi interactions. High resolution contact mapping was conducted using multiple techniques (missing base contact probing, methylation protection, base modification, and hydroxyl radical footprinting), and the electrophoretic separation of nucleoprotein complexes allowed us to discriminate between contact patterns produced by pi monomers and dimers. We also isolated iteron mutants that affected the binding of pi monomers (only) or both monomers and dimers. The mutational studies and footprinting analyses revealed that, when binding DNA, pi monomers interact with nucleotides spanning the entire length of the iteron. In contrast, pi dimers interact with only the left half of the iteron; however, the retained interactions are strikingly similar to those seen with monomers. These results support a model in which Rep protein dimerization disturbs one of two DNA binding domains important for monomer/iteron interaction; the dimer/iteron interaction utilizes only one DNA binding domain.
Kunnimalaiyaan
Selvi
S
Department of Bacteriology, University of Wisconsin, Madison, Wisconsin 53706, USA.
Krüger
Ricardo
R
Ross
Wilma
W
Rakowski
Sheryl A
SA
Filutowicz
Marcin
M
D001483:Base Sequence; D004247:DNA / Q000737:chemistry; D018983:DNA Footprinting; D004265:DNA Helicases / Q000378:metabolism / Q000502:physiology*; D019175:DNA Methylation; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins / Q000378:metabolism / Q000502:physiology*; D019281:Dimerization; D004926:Escherichia coli / Q000378:metabolism; D017665:Hydroxyl Radical; D008957:Models, Genetic; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D018741:Replication Origin; D015534:Trans-Activators / Q000378:metabolism / Q000502:physiology*
D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D015534:Trans-Activators; C045264:replication initiator protein; D017665:Hydroxyl Radical; D004247:DNA; D004265:DNA Helicases