M. Llosa et al., FUNCTIONAL DOMAINS IN PROTEIN TRWC OF PLASMID R388 - DISSECTED DNA STRAND TRANSFERASE AND DNA HELICASE ACTIVITIES RECONSTITUTE PROTEIN FUNCTION, Journal of Molecular Biology, 264(1), 1996, pp. 56-67
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