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 termination 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 relaxation domain of the protein. Simultaneous mutation of both Y18 and Y26 was required to abolish in vitro cleavage and strand-transfer reactions catalyzed by 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, abolished nic-cleavage of a supercoiled DNA containing the R388 origin of transfer (oriT). These data allowed the construction of a model for conjugative DNA processing in which Y18 specifically catalyzes the initial cleavage reaction, while Y26 is used for the second strand-transfer reaction, which terminates conjugation. The model suggests a control mechanism that can be effective at each conjugative replication cycle.
Grandoso
G
G
Departamento de Biología Molecular, Universidad de Cantabria, C. Cardenal Herrera Oria s/n, Santander, 39011, Spain.
Avila
P
P
Cayón
A
A
Hernando
M A
MA
Llosa
M
M
de la Cruz
F
F
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D001665:Binding Sites; D002384:Catalysis; D003227:Conjugation, Genetic / Q000235:genetics*; D018736:DNA Adducts / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004254:DNA Nucleotidyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004278:DNA, Superhelical / Q000235:genetics; D010450:Endopeptidases / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D019426:Integrases; D008957:Models, Genetic; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D009838:Oligodeoxyribonucleotides / Q000235:genetics; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics*; D045522:Recombinases; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics; D016415:Sequence Alignment; D013997:Time Factors; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism*
D018736:DNA Adducts; D004278:DNA, Superhelical; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments; D045522:Recombinases; D014443:Tyrosine; D004254:DNA Nucleotidyltransferases; D019426:Integrases; C115006:integron integrase IntI1; D010450:Endopeptidases