Athanasia Varsaki, Gabriel Moncalián, Maria del Pilar Garcillán-Barcia, Constantin Drainas, and Fernando de la Cruz*
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 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 a conserved arginine (R13 in MbeC) which was mutated to alanine. The mutant MbeC(R13A) was unable to bind either ssDNA 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. 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 so far.
http://dx.doi.org/10.1128/JB.01342-08
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 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 a conserved arginine (R13 in MbeC) which was mutated to alanine. The mutant MbeC(R13A) was unable to bind either ssDNA 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. 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 so far.
http://dx.doi.org/10.1128/JB.01342-08
