Analysis of DNA processing reactions in bacterial conjugation by using suicide oligonucleotides. The EMBO Journal

Blanca Gonzalez-Perez, María Lucas, Leonie A Cooke, Joseph S Vyle, Fernando de la Cruz and Gabriel Moncalián
Departamento de Biología Molecular (Universidad de Cantabria) and Instituto de Biomedicina y Biotecnología de Cantabria (CSIC-UC-IDICAN), Santander, Spain2 School of Chemistry and Chemical Engineering, The Queen's University of Belfast, Belfast, UKTo whom correspondence should be addressedFernando de la Cruz, Departamento de Biologia Molecular, Instituto de Biomedicina y Biotecnología de Cantabria (CSIC), Universidad de Cantabria, Cardenal Herrera Oria s/n, Santander 39011, Spain. Tel.: +34 94 2201 942; Fax: +34 94 2201 945; E-mail: delacruz@unican.es Received 9 March 2007; Accepted 26 June 2007; Published online 26 July 2007.
Abstract
Protein TrwC is the conjugative relaxase responsible for DNA processing in plasmid R388 bacterial conjugation. TrwC has two catalytic tyrosines, Y18 and Y26, both able to carry out cleavage reactions using unmodified oligonucleotide substrates. Suicide substrates containing a 3'-S-phosphorothiolate linkage at the cleavage site displaced TrwC reaction towards covalent adducts and thereby enabled intermediate steps in relaxase reactions to be investigated. Two distinct covalent TrwC–oligonucleotide complexes could be separated from noncovalently bound protein by SDS–PAGE. As observed by mass spectrometry, one complex contained a single, cleaved oligonucleotide bound to Y18, whereas the other contained two cleaved oligonucleotides, bound to Y18 and Y26. Analysis of the cleavage reaction using suicide substrates and Y18F or Y26F mutants showed that efficient Y26 cleavage only occurs after Y18 cleavage. Strand-transfer reactions carried out with the isolated Y18–DNA complex allowed the assignment of specific roles to each tyrosine. Thus, only Y18 was used for initiation. Y26 was specifically used in the second transesterification that leads to strand transfer, thus catalyzing the termination reaction that occurs in the recipient cell.
Keywords: 3'-S-phosphorothiolate-containing oligonucleotides, bacterial conjugation, relaxase, transesterification