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. 2011 Mar;79(6):1670–1683. doi: 10.1111/j.1365-2958.2011.07553.x

Fig. 5.

Fig. 5

Site- and strand-specific relaxation of the 89K oriT region.

A. Physical map of the pMD-oriT plasmid, which contains the oriT region of 89K and serves as substrate DNA in the relaxation analysis. The putative oriT nick site (nic) is indicated by a vertical arrow.

B. Equivalent substrate pMD-oriT plasmid was incubated with (+) or without (–) the proteins of interest. Reaction products were analysed by standard agarose gel electrophoresis (0.9%). OC, open circular plasmid DNA; CCC, covalently closed circular plasmid DNA.

C. Schematic representation of the expected single-strand species generated by a site-specific nick (arrow) on endonuclease-linearized pMD-oriT DNA. The size of each ssDNA fragment is shown.

D. pMD-oriT was linearized with either ScaI or NdeI, and the relaxation mixtures were analysed on a 0.9% alkaline agarose gel. Lanes: 1, ScaI-linearized pMD-oriT with MobA89K and MobC89K; 2, NdeI-linearized pMD-oriT with MobA89K and MobC89K; 3, ScaI-linearized pMD-oriT with MobA89KN258 and MobC89K; 4, NdeI-linearized pMD-oriT with MobA89K and MobC89K. The 1 kb DNA ladder marker is shown to the right (M).