DNA recognition by F factor TraI36: Highly sequence-specific binding of single-stranded DNA

The TraI protein has two essential roles in transfer of conjugative plasmid F Factor. As part of a complex of DNA-binding proteins, TraI introduces a site- and strand-specific nick at the plasmid origin of transfer (oriT), cu tting the DNA strand that is transferred to the recipient cell. TraI also a cts as a helicase, presumably unwinding the plasmid strands prior to transf er. As an essential feature of its nicking activity, TraI is capable of bin ding and cleaving single-stranded DNA oligonucleotides containing an oriT s equence. The specificity of TraI DNA recognition was examined by measuring the binding of oriT oligonucleotide variants to TraI36, a 36-kD amino-termi nal domain of TraI that retains the sequence-specific nucleolytic activity. TraI36 recognition is highly sequence-specific for an 11-base region of or iT, with single base changes reducing affinity by as much as 8000-fold. The binding data correlate with plasmid mobilization efficiencies: plasmids co ntaining sequences bound with lower affinities by TraI36 are transferred be tween cells at reduced frequencies. In addition to the requirement for high affinity binding to oriT, efficient in vitro nicking and in vivo plasmid m obilization requires a pyrimidine immediately 5' of the nick site. The high sequence specificity of TraI single-stranded DNA recognition suggests that despite its recognition of single-stranded DNA, TraI is capable of playing a major regulatory role in initiation and/or termination of plasmid transf er.