title: Analysis of ColE1 MbeC unveils an extended ribbon-helix-helix family of nicking accessory proteins.
abstract: MbeC is a 13-kDa ColE1-encoded protein required for efficient mobilization of ColE1, a plasmid widely used in cloning vector technology. MbeC protein was purified and used for in vitro DNA binding, which showed that it binds specifically double-stranded DNA (dsDNA) containing the ColE1 oriT. Amino acid sequence comparison and secondary structure prediction imply that MbeC is related to the ribbon-helix-helix (RHH) protein family. Alignment with RHH members pointed to a conserved arginine (R13 in MbeC) that was mutated to alanine. The mutant MbeC(R13A) was unable to bind either single-stranded DNA or dsDNA. Limited proteolysis fragmented MbeC in two stable folding domains: the N-terminal domain, which contains the RHH motif, and the C-terminal domain, which comprises a signature shared by nicking accessory proteins. The results indicate that MbeC plays a similar role in conjugation as TraY and TrwA of plasmids F and R388, respectively. Thus, it appears that an extended, possibly universal mechanism of DNA conjugative processing exists, in which oriT-processing is carried out by relaxases assisted by homologous nicking accessory proteins. This mechanism seems to be shared by all major conjugative systems analyzed thus far.
authors.affiliation: Department of Chemistry, University of Ioannina, Greece.
authors.forename: Athanasia
authors.initials: A
authors.lastname: Varsaki
authors.forename: Gabriel
authors.initials: G
authors.lastname: Moncalián
authors.forename: Maria del Pilar
authors.initials: Mdel P
authors.lastname: Garcillán-Barcia
authors.forename: Constantin
authors.initials: C
authors.lastname: Drainas
authors.forename: Fernando
authors.initials: F
authors.lastname: de la Cruz
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D003086:Bacteriocin Plasmids / Q000235:genetics*; D018387:Codon, Initiator; D003227:Conjugation, Genetic; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D018741:Replication Origin
chemical_list: D001426:Bacterial Proteins; D018387:Codon, Initiator; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C083180:Mob protein, Bacteria