title: Completing the specificity swap: Single-stranded DNA recognition by F and R100 TraI relaxase domains.
abstract: During conjugative plasmid transfer, one plasmid strand is cleaved and transported to the recipient bacterium. For F and related plasmids, TraI contains the relaxase or nickase activity that cleaves the plasmid DNA strand. F TraI36, the F TraI relaxase domain, binds a single-stranded origin of transfer (oriT) DNA sequence with high affinity and sequence specificity. The TraI36 domain from plasmid R100 shares 91% amino acid sequence identity with F TraI36, but its oriT DNA binding site differs by two of eleven bases. Both proteins readily distinguish between F and R100 binding sites. In earlier work, two amino acid substitutions in the DNA binding cleft were shown to be sufficient to change the R100 TraI36 DNA-binding specificity to that of F TraI36. In contrast, three substitutions could make F TraI36 more "R100-like", but failed to completely alter the specificity. Here we identify one additional amino acid substitution that completes the specificity swap from F to R100. To our surprise, adding further substitutions from R100 to the F background were detrimental to binding instead of being neutral, indicating that their effects were influenced by their structural context. These results underscore the complex and subtle nature of DNA recognition by relaxases and have implications for the evolution of relaxase binding sites and oriT sequences.
authors.affiliation: Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218, USA.
authors.forename: Kip E
authors.initials: KE
authors.lastname: Guja
authors.affiliation: Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218, USA. Electronic address: joel@jhu.edu.
authors.forename: Joel F
authors.initials: JF
authors.lastname: Schildbach
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001426:Bacterial Proteins; D001483:Base Sequence; D020134:Catalytic Domain; D004265:DNA Helicases / Q000235:genetics*; D004722:Endoribonucleases / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics*; D007700:Kinetics; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D017385:Sequence Homology; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D004722:Endoribonucleases; C027093:TraI protein, E coli; D004265:DNA Helicases
keywords: Conjugation; Nickase; Plasmid; Relaxase; Specificity