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Journal of Bacteriology, March 2009, p. 1446-1455, Vol. 191, No. 5
0021-9193/09/$08.00+0     doi:10.1128/JB.01342-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Analysis of ColE1 MbeC Unveils an Extended Ribbon-Helix-Helix Family of Nicking Accessory Proteins{triangledown}

Athanasia Varsaki ,1,{dagger},{ddagger} Gabriel Moncalián,2,{dagger} Maria del Pilar Garcillán-Barcia,2 Constantin Drainas,1 and Fernando de la Cruz2*

Sector of Organic Chemistry and Biochemistry, Department of Chemistry, University of Ioannina, Ioannina, Greece,1 Departamento de Biología Molecular, Universidad de Cantabria, and Instituto de Biomedicina y Biotecnología de Cantabria (CSIC-UC-IDICAN), Santander, Spain2

Received 26 September 2008/ Accepted 17 December 2008

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.

* Corresponding author. Mailing address: Departamento de Biología Molecular, Universidad de Cantabria, C/Herrera Oria s/n, 39011 Santander, Spain. Phone: 34 942 201942. Fax: 34 942 201945. E-mail: delacruz{at}unican.es

{triangledown} Published ahead of print on 29 December 2008.

{dagger} A.V. and G.M. contributed equally to this study.

{ddagger} Present address: IBBS Biophysics Laboratories, School of Biological Sciences, University of Portsmouth, Portsmouth, United Kingdom.

Journal of Bacteriology, March 2009, p. 1446-1455, Vol. 191, No. 5
0021-9193/09/$08.00+0     doi:10.1128/JB.01342-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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