Isomerization and apparent DNA bending by pi, the replication protein of plasmid R6K.
Plasmid R6K-encoded pi protein has multiple regulatory functions in replication and transcription. These functions rely, in part, on a complex set of interactions between monomers and dimers of the protein and distinct DNA targets, the direct and inverted repeats (DRs, IRs). In the work described here, we examine the isomerization and DNA bending properties of pi using electrophoretic mobility shift assays and circular permutation assays. Our data suggest that pi dimers can bend IRs, and dimer subunits seem to readily associate in head-to-head and head-to-tail fashion. The ability of pi to bend DRs is also reexamined using techniques that allow us to discriminate between bending induced by its different isomeric forms. We find that both monomers and dimers bend a single DR to similar degrees while results with 2DRs are more complex. The significance of the bending data in regard to a possible mechanism for replication initiation by pi protein is discussed.
Krüger
Ricardo
R
Universidade Católica de Brasi;lia, Campus II, SGAN 916, Módulo B, W5 Norte, Brasília, Brazil.
Rakowski
Sheryl A
SA
Filutowicz
Marcin
M
D004265:DNA Helicases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004269:DNA, Bacterial / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D015534:Trans-Activators; C045264:replication initiator protein; D004265:DNA Helicases