Structural changes in RepA, a plasmid replication initiator, upon binding to origin DNA.
RepA protein is the DNA replication initiator of the Pseudomonas plasmid pPS10. RepA dimers bind to an inversely repeated operator sequence in repA promoter, thus repressing its own synthesis, whereas monomers bind to four directly repeated sequences (iterons) to initiate DNA replication. We had proposed previously that RepA is composed of two winged-helix (WH) domains, a structural unit also present in eukaryotic and archaeal initiators. To bind to the whole iteron sequence through both domains, RepA should couple monomerization to a conformational change in the N-terminal WH, which includes a leucine zipper-like sequence motif. We show for the first time that, by itself, binding to iteron DNA in vitro dissociates RepA dimers into monomers and alters RepA conformation, suggesting an allosteric effect. Furthermore, we also show that similar changes in RepA are promoted by mutations that substitute two Leu residues of the putative leucine zipper by Ala, destabilizing the hydrophobic core of the first WH. We propose that this mutant (RepA-2L2A) resembles a transient folding intermediate in the pathway leading to active monomers. These findings, together with the known activation of other Rep-type proteins by chaperones, are relevant to understand the molecular basis of plasmid DNA replication initiation.
Díaz-López
Teresa
T
Department of Molecular Microbiology, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Cientificas (CSIC), C/Velázquez, 144, 28006 Madrid, Spain.
Lages-Gonzalo
Marta
M
Serrano-López
Ana
A
Alfonso
Carlos
C
Rivas
Germán
G
Díaz-Orejas
Ramón
R
Giraldo
Rafael
R
D020816:Amino Acid Motifs; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism*; D004265:DNA Helicases; D004268:DNA-Binding Proteins; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D006639:Histidine / Q000737:chemistry; D007700:Kinetics; D007930:Leucine / Q000737:chemistry; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010957:Plasmids / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D011550:Pseudomonas aeruginosa / Q000235:genetics* / Q000378:metabolism; D018741:Replication Origin; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013997:Time Factors; D015534:Trans-Activators; D014461:Ultracentrifugation
D004268:DNA-Binding Proteins; D011506:Proteins; D015534:Trans-Activators; C045264:replication initiator protein; D006639:Histidine; D004247:DNA; D004265:DNA Helicases; D007930:Leucine