TrwC is a bifunctional enzyme that displays two biochemical activities essential for plasmid R388 conjugation: oriT-specific DNA strand-transferase and DNA helicase activities. We overproduced and purified different segments of the protein allowing us to map the relaxase and DNA helicase activities to separate regions of the protein. A peptide comprising the N-terminal 275 amino acid residues of the protein was able to catalyze DNA cleavage and strand-transfer reactions when using oligonucleotides encompassing the nic site, although a longer fragment of TrwC (348 amino acid residues) was required to produce the nick on a supercoiled double-stranded DNA substrate. The segment of the protein between amino acid residues 192 and 966 contained the ATPase and DNA helicase activities, while a peptide consisting of amino acid residues 346 to 966 lost both activities. The dimerization region lay in the 495 C-terminal amino acid residues. Two peptides containing the DNA strand-transferase and DNA helicase activities, respectively, could functionally substitute for TrwC in R388 conjugation although at a 10,000-fold lower efficiency. Thus, integrity of the covalent structure of the protein was required for efficient DNA transfer. It can be assumed that the covalent linkage increases the efficiency of conjugation by increasing the effective concentration of one component (presumably the DNA helicase) at its site of action.