title: Nicking activity of the pMV158 MobM relaxase on cognate and heterologous origins of transfer.
abstract: The MobM relaxase (494 amino acids) encoded by the promiscuous streptococcal plasmid pMV158 recognizes the plasmid origin of transfer, oriTpMV158, and converts supercoiled pMV158 DNA into relaxed molecules by cleavage of the phosphodiester bond of a specific dinucleotide within the sequence 5'-GTGTG/TT-3' ("/" being the nick site). After cleavage, the protein remains stably bound to the 5'-end of the nick site. Band-shift assays with single-stranded oligonucleotides and size-exclusion chromatography allowed us to show that MobM was able to generate specific complexes with one of the inverted repeats of the oriTpMV158, presumably extruded as stem-loop structure. A number of tests have been performed to attain a better characterization of the nicking activity of MobM and its linkage with its target DNA. The optimal pH for DNA relaxation was found to be 6.5. Upon nicking, gel retardation assays showed that MobM formed stable complexes with its target DNA. Moreover, MobM bound to relaxed pMV158 molecules were visualized by electron microscopy. The staphylococcal plasmids pUB110 and pE194, and the streptococcal plasmid pDL287 harbour putative oriTs and may encode Mob proteins homologous to MobM. The oriTpUB110, oriTpDL287, and oriTpE194 sequences share 100%, 70%, and 67% (in a 43-nucleotide stretch and allowing a 3-bp gap) identity to oriTpMV158, respectively. Nicking assays using supercoiled DNAs from pUB110, pDL287, and pE194 showed that MobM was able to relax, to differing degrees, all plasmid DNAs. Our results suggest that cross-recognition of heterologous oriTs by Mob proteins could play an important role in the plasmid spreading between bacteria.
authors.affiliation: Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Ramiro de Maeztu 9, 28040 Madrid, Spain.
authors.forename: Cris
authors.initials: C
authors.lastname: Fernández-López
authors.forename: Fabián
authors.initials: F
authors.lastname: Lorenzo-Díaz
authors.forename: Rosa
authors.initials: R
authors.lastname: Pérez-Luque
authors.forename: Lorena
authors.initials: L
authors.lastname: Rodríguez-González
authors.forename: Roeland
authors.initials: R
authors.lastname: Boer
authors.forename: Rudi
authors.initials: R
authors.lastname: Lurz
authors.forename: Alicia
authors.initials: A
authors.lastname: Bravo
authors.forename: Miquel
authors.initials: M
authors.lastname: Coll
authors.forename: Manuel
authors.initials: M
authors.lastname: Espinosa
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001665:Binding Sites; D003227:Conjugation, Genetic; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004278:DNA, Superhelical / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D004706:Endodeoxyribonucleases / Q000235:genetics* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D055029:Inverted Repeat Sequences; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011485:Protein Binding; D012689:Sequence Homology, Nucleic Acid; D013296:Streptococcus pneumoniae / Q000201:enzymology / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004278:DNA, Superhelical; D004706:Endodeoxyribonucleases; C120220:MobM protein, bacteria