FUNCTIONAL DOMAINS IN PROTEIN TRWC OF PLASMID R388 - DISSECTED DNA STRAND TRANSFERASE AND DNA HELICASE ACTIVITIES RECONSTITUTE PROTEIN FUNCTION

TrwC is a bifunctional enzyme that displays two biochemical activities essential for plasmid R388 conjugation: oriT-specific DNA strand-tran sferase and DNA helicase activities. We overproduced and purified diff erent segments of the protein allowing us to map the relaxase and DNA helicase activities to separate regions of the protein. A peptide comp rising the N-terminal 275 amino acid residues of the protein was able to catalyze DNA cleavage and strand-transfer reactions when using olig onucleotides encompassing the nic site, although a longer fragment of TrwC (348 amino acid residues) was required to produce the nick on a s upercoiled double-stranded DNA substrate. The segment of the protein b etween amino acid residues 192 and 966 contained the ATPase and DNA he licase activities, while a peptide consisting of amino acid residues 3 46 to 966 lost both activities. The dimerization region lay in the 495 C-terminal amino acid residues. Two peptides containing the DNA stran d-transferase and DNA helicase activities, respectively, could functio nally substitute for TrwC in R388 conjugation although at a 10,000-fol d lower efficiency. Thus, integrity of the covalent structure of the p rotein was required for efficient DNA transfer. It can be assumed that the covalent linkage increases the efficiency of conjugation by incre asing the effective concentration of one component (presumably the DNA helicase) at its site of action. (C) 1996 Academic Press Limited