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Analysis of ColE1 MbeC unveils an extended ribbon-helix-helix family of nicking accessory proteins

AuthorsVarsaki, Athanasia ; Moncalián, Gabriel ; Garcillán-Barcia, M. Pilar; Drainas, Constantin; Cruz, Fernando de la
Issue Date2009
PublisherAmerican Society for Microbiology
CitationJournal of Bacteriology 191(5): 1446-1455 (2009)
AbstractMbeC is a 13-kDa ColEl-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 ColEl 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. Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Identifiersdoi: 10.1128/JB.01342-08
issn: 0021-9193
Appears in Collections:(IBBTEC) Artículos
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