Published online 17 January 2006
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Examination of an inverted repeat within the F factor origin of transfer: context dependence of F TraI relaxase DNA specificity
Department of Biology, The Johns Hopkins University 3400 North Charles Street, Baltimore, MD 21218, USA
*To whom correspondence should be addressed. Tel: +1 410 516 0176; Fax: +1 410 516 5213; Email: joel{at}jhu.edu
Received October 4, 2005. Revised December 30, 2005. Accepted December 30, 2005.
Prior to conjugative transfer of plasmids, one plasmid strand is cleaved in a site- and strand-specific manner by an enzyme called a relaxase or nickase. In F and related plasmids, an inverted repeat is located near the plasmid strand cleavage site, and others have proposed that the ability of this sequence to form a hairpin when in single-stranded form is important for transfer. Substitutions were introduced into a cloned F oriT region and their effects on plasmid transfer were assessed. For those substitutions that substantially reduced transfer, the results generally correlated with effects on in vitro binding of oligonucleotides to the F TraI relaxase domain rather than with predicted effects on hairpin formation. One substitution shown previously to dramatically reduce both plasmid transfer and in vitro binding to a 17-base oligonucleotide had little apparent effect on binding to a 30-base oligonucleotide that contained the hairpin region. Results from subsequent experiments strongly suggest that the relaxase domain can bind to hairpin oligonucleotides in two distinct manners with different sequence specificities, and that the protein binds the oligonucleotides at the same or overlapping sites.
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