The relaxase (TraI) of plasmid RP4 (IncPalpha) plays a key role in initiation and termination of transfer DNA replication during conjugative transmission of the plasmid. TraI functions as a DNA strand transferase that cleaves a unique phosphodiester bond at nic of the transfer origin. The cleavage reaction consists in a reversible transesterification that leads to transfer of the 5' phosphoryl at nic to the hydroxyl group of TraI Tyr-22. Hence, cleavage results in the covalent attachment of TraI to the 5' terminus of the plasmid strand destined for transfer. To investigate the protein's ability to function in a "second cleavage" reaction proposed to terminate rolling circle mode transfer DNA replication, single-stranded oligonucleotides containing the nic region were immobilized at their 3' ends on magnetic beads and cleaved by TraI. The resulting covalent TraI-oligonucleotide adducts were active in the joining reaction but unable to cleave oligonucleotides containing an intact nic region, indicating that second cleavage probably requires a TraI dimer, since a monomer is insufficient. The covalently attached oligonucleotide determines the affinity of the relaxase for the 3' terminus of the T-strand. To further the biochemical characterization of TraI-catalyzed reactions, we used specific TraI mutants, showing that amino acid residues in each relaxase motif are involved in substrate binding. To uncouple substrate binding and cleaving-joining, we applied partially biotinylated TraI mutant proteins that were immobilized to magnetic beads. Using this approach we could demonstrate that tight DNA substrate binding and cleaving-joining are independent processes. Enhanced topoisomerase activity of some TraI mutants was correlated with low specific substrate binding affinity in conjunction with high cleaving-joining activity.