Negative regulation of pPS10 plasmid replication: origin pairing by zipping-up DNA-bound RepA monomers.
In many plasmid replicons of gram-negative bacteria, Rep protein dimers are transcriptional self-repressors of their genes, whereas monomers are initiators of DNA replication. Switching between both functions implies conformational remodelling of Rep, and is promoted by Rep binding to the origin DNA repeats (iterons) or chaperones. Rep proteins play another key role: they bridge together two iteron DNA stretches, found either on the same or on different plasmid molecules. These so-called, respectively, 'looped' and 'handcuffed' complexes are thought to be negative regulators of plasmid replication. Although evidence for Rep-dependent plasmid handcuffing has been found in a number of replicons, the structure of these Rep-DNA assemblies is still unknown. Here, by a combination of proteomics, electron microscopy, genetic analysis and modelling, we provide insight on a possible three-dimensional structure for two handcuffed arrays of the iterons found at the origin of pPS10 replicon. These are brought together in parallel register by zipping-up DNA-bound RepA monomers. We also present evidence for a distinct role of RepA dimers in DNA looping. This work defines a new regulatory interface in Rep proteins.
Gasset-Rosa
Fátima
F
Department of Molecular Microbiology, Centro de Investigaciones Biológicas-CSIC, Madrid, Spain.
Díaz-López
Teresa
T
Lurz
Rudi
R
Prieto
Alicia
A
Fernández-Tresguerres
M Elena
ME
Giraldo
Rafael
R
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004265:DNA Helicases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004261:DNA Replication; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D021621:Imaging, Three-Dimensional; D046911:Macromolecular Substances / Q000737:chemistry; D046529:Microscopy, Electron, Transmission; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D009875:Operator Regions, Genetic; D010449:Peptide Mapping; D010957:Plasmids / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure; D017434:Protein Structure, Tertiary; D040901:Proteomics; D011550:Pseudomonas aeruginosa / Q000235:genetics*; D018741:Replication Origin; D015534:Trans-Activators / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D015534:Trans-Activators; C045264:replication initiator protein; D004265:DNA Helicases