title: An intrastrand three-DNA-base interaction is a key specificity determinant of F transfer initiation and of F TraI relaxase DNA recognition and cleavage.
abstract: Bacterial conjugation, transfer of a single conjugative plasmid strand between bacteria, diversifies prokaryotic genomes and disseminates antibiotic resistance genes. As a prerequisite for transfer, plasmid-encoded relaxases bind to and cleave the transferred plasmid strand with sequence specificity. The crystal structure of the F TraI relaxase domain with bound single-stranded DNA suggests binding specificity is partly determined by an intrastrand three-way base-pairing interaction. We showed previously that single substitutions for the three interacting bases could significantly reduce binding. Here we examine the effect of single and double base substitutions at these positions on plasmid mobilization. Many substitutions reduce transfer, although the detrimental effects of some substitutions can be partially overcome by substitutions at a second site. We measured the affinity of the F TraI relaxase domain for several DNA sequence variants. While reduced transfer generally correlates with reduced binding affinity, some oriT variants transfer with an efficiency different than expected from their binding affinities, indicating ssDNA binding and cleavage do not correlate absolutely. Oligonucleotide cleavage assay results suggest the essential function of the three-base interaction may be to position the scissile phosphate for cleavage, rather than to directly contribute to binding affinity.
authors.affiliation: Department of Biology, Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218, USA.
authors.forename: Katherine
authors.initials: K
authors.lastname: Hekman
authors.forename: Kip
authors.initials: K
authors.lastname: Guja
authors.forename: Chris
authors.initials: C
authors.lastname: Larkin
authors.forename: Joel F
authors.initials: JF
authors.lastname: Schildbach
mesh_terms: D020029:Base Pairing; D003227:Conjugation, Genetic; D053837:DNA Cleavage; D004265:DNA Helicases / Q000737:chemistry* / Q000378:metabolism; D004254:DNA Nucleotidyltransferases / Q000737:chemistry* / Q000378:metabolism; D004269:DNA, Bacterial / Q000737:chemistry* / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000737:chemistry* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D005144:F Factor / Q000235:genetics*; D008958:Models, Molecular; D011485:Protein Binding
chemical_list: D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D029968:Escherichia coli Proteins; D004254:DNA Nucleotidyltransferases; C110340:DNA relaxase; D004706:Endodeoxyribonucleases; C027093:TraI protein, E coli; D004265:DNA Helicases