Two active-site tyrosyl residues of protein TrwC act sequentially at the origin of transfer during plasmid R388 conjugation

Protein TrwC is the relaxase-helicase responsible for the initiation and te rmination reactions of DNA processing during plasmid R388 conjugation. Site -directed mutagenesis was used to change to phenylalanine each of a set of four conserved tyrosyl residues in the sequence of the N-terminal relaxatio n domain of the protein. Simultaneous mutation of both Y18 and Y26 was requ ired to abolish in vitro cleavage and strand-transfer reactions catalyzed b y protein TrwC on oligonucleotides containing the nic site. Thus, both Y18 and Y26 could be involved independently in the formation of oligonucleotide -protein covalent complexes that constitute presumed intermediates of these reactions. This hypothesis was confirmed by the observation of Y18 and Y26 -specific peptide-oligonucleotide adducts after protease digestion of TrwC and mutant derivatives. Finally mutation Y18F, but not mutation Y26F, aboli shed nic-cleavage of a supercoiled DNA containing the R388 origin of transf er (oriT). These data allowed the construction of a model for conjugative D NA processing in which Y18 specifically catalyzes the initial cleavage reac tion, while Y26 is used for the second strand-transfer reaction, which term inates conjugation. The model suggests a control mechanism that can be effe ctive at each conjugative replication cycle. (C) 2000 Academic Press.