Relaxase (TraI) of IncPα Plasmid RP4 Catalyzes a Site-Specific Cleaving-Joining Reaction of Single-Stranded DNA

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 90; no. 7; pp. 2925 - 2929
• Main Authors: Pansegrau, Werner, Schroder, Werner, Lanka, Erich
• Format: Journal Article
• Language: English
• Published: Washington, DC National Academy of Sciences of the United States of America 01.04.1993; National Acad Sciences
• Subjects: Adducts; Amino Acid Sequence; Amino acids; Bacteriology; Base Sequence; Binding Sites; Biological and medical sciences; DNA; DNA Helicases - genetics; DNA Helicases - metabolism; DNA Replication; DNA, Single-Stranded - metabolism; Endonucleases - metabolism; Escherichia coli Proteins; Fundamental and applied biological sciences. Psychology; Gels; Genetics; Kinetics; Microbiology; Models, Structural; Molecular Sequence Data; Mutagenesis, Site-Directed; Nucleotides; Oligodeoxyribonucleotides; Oligodeoxyribonucleotides - metabolism; Oligonucleotides; Peptide Mapping; Plasmids; Product labeling; Protein Conformation; Substrate Specificity; Teeth; Plasmid; Site specificity; Enzyme; Bacteria; Evolution; Molecular interaction; Replication; Single stranded DNA; Conjugation; In vitro; Mechanism; Genetic transfer
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.90.7.2925

Conjugative DNA transfer of the self-transmissible broad-host-range plasmid RP4 is initiated by strand- and site-specific cleavage at the nick site (nic) of the transfer origin (oriT). Cleavage results in covalent attachment of the plasmid-encoded relaxase (TraI) to the 5'-terminal 2'-deoxycytidine residue at nic. We demonstrate that Tyr22is the center of the catalytic site of TraI, mediating cleavage via formation of a phosphodiester between the DNA 5' phosphoryl and the aromatic hydroxyl group. The specificity of cleavage seen with form I oriT DNA was verified with short oligodeoxyribonucleotides embracing the nick region. The reaction requires TraI and Mg2+but is independent of the relaxosome component TraJ. Cleavage produces one oligonucleotide fragment with a free 3' hydroxyl, the other part forms a covalent TraI-oligonucleotide adduct. Like nicking of form I oriT DNA, TraI-catalyzed oligonucleotide cleavage reaches an equilibrium when about 30% of the input TraI exists as a covalent protein-DNA complex. In the presence of two differently sized oligonucleotides, defined hybrid oligonucleotides are produced, demonstrating that TraI catalyzes recombination of two single strands at nic. This finding shows that TraI possesses cleaving-joining activity resembling that of a type I topoisomerase. Reactions are dependent on the sequence of the 3'-terminal 6 nucleotides adjacent to nic. Only certain base changes in a few positions are tolerated, whereas the sequence of the 5' terminal nucleotides apparently is irrelevant for recognition by TraI. The reactions described here further support the hypothesis that DNA transfer via conjugation involves a rolling circle-like mechanism which generates the immigrant single strand while DNA-bound TraI protein scans for the occurrence of a second cleavage site at the donor-recipient interface.