FIG. 2.

Intracellular oriT cleavage by TraI and TraI::i31 mutants. (A) Autoradiogram of runoff DNA synthesis products measuring TraI-catalyzed cleavage activity. E. coli AG1 strains harboring pLOW2traM0oriT and TraI-expressing plasmids as shown above were harvested after subculture in fresh medium and IPTG induction. All reaction mixtures contained equivalent cell mass and 2 ng of a purified DNA standard (std). The numbers of viable cells in the reaction mixtures resolved in lanes 1 to 16 were 2, 0.27, 0.54, 0.16, 0.32, 0.25, 0.5, 0.27, 0.54, 0.3, 0.6, 1.5, 3, 0.7, 1.4, and 0.81 (10⁷ CFU), respectively. The source of products that are terminated within the A stretch proximal to nic is not known, but these and shorter extension products are consistently observed when high concentrations of cells are present in reaction mixtures. Also typical for this assay is the apparent double band at nic, which is consistent with the terminal transferase activity of the polymerase used in vitro. (B) Standard DNA product after a threefold- longer exposure time. +, present; −, negative control reaction.