Figure 3.

The TraI transesterification reaction is not efficient. About 20 nM or 1 µM of TraI relaxase domain and 20 nM of 30-base F oriT hairpin (top gel) or F oriT no hairpin oligonucleotide (bottom gel) 5′-labeled with a Cy5 fluorophore were incubated in reaction buffer for 1 h at 37°C. About 1 µM of TraI Y16F was added where indicated and the incubation was continued for an additional 1 h. Reactions were terminated with proteinase K. Cleaved ssDNA is 3 nt shorter and therefore migrates farther. Oligonucleotide sequences are given in Table 1 and Supplementary Table SII.