Protein motion from non-specific to specific DNA by three-dimensional routes aided by supercoiling

Abstract

DNA-binding proteins are generally thought to locate their target sites by first associating with the DNA at random and then translocating to the specific site by one-dimensional (1D) diffusion along the DNA. We report here that non-specific DNA conveys proteins to their target sites just as well when held near the target by catenation as when co-linear with the target. Hence, contrary to the prevalent view, proteins move from random to specific sites primarily by three-dimensional (3D) rather than 1D pathways, by multiple dissociation/re-association events within a single DNA molecule. We also uncover a role for DNA supercoiling in target-site location. Proteins find their sites more readily in supercoiled than in relaxed DNA, again indicating 3D rather than 1D routes.

Fig. 1. DNA substrates. (A) The plasmid pDG2 contains recognition sites for EcoRV and HindIII, at the positions marked E and H, respectively, and two res sites from the transposon Tn21 (triangles, marked R). The 346 bp segment of DNA between the res sites also contains single sites for BsaAI, ClaI, Eco47III, PstI and XhoI. (B) Recombin ation at the res sites in pDG2, by Tn21 resolvase, generates a catenane with two interlinked rings of 3120 and 346 bp; the sites for EcoRV (marked E), BsaAI, ClaI, Eco47III, PstI and XhoI are in the 346 bp circle and that for HindIII (H) in the 3120 bp circle. (C) The 346 bp minicircle is liberated from the 3120 bp ring by cleaving the latter with HindIII.

Fig. 2. Supercoiled DNA. The reactions contained two DNA substrates, both at 25 nM, and 0.2 nM EcoRV endonuclease in reaction buffer at 37°C. Samples were removed at various times after adding the EcoRV and analysed as in Materials and methods for the extent of cleavage of each DNA. Data from four repeats are shown: circles, squares, triangles and diamonds mark the individual experiments. The two substrates in each reaction are as indicated above the respective panel: (A) supercoiled plasmid (filled data points) and minicircle (unfilled data points); (B) supercoiled catenane (filled points) and minicircle (unfilled points); and (C) plasmid (filled points) and catenane (unfilled points), both supercoiled.

Fig. 3. Potential effects of supercoiling on target-site location. The upper pictures show the relaxed and the supercoiled forms of a plasmid with a single recognition site for EcoRV (marked E). Two landing sites for the initial binding of the protein are marked LS1 and LS2: the 1D distance along the DNA between E and LS1 is shorter than that between E and LS2. In the relaxed DNA, the mean 3D distance between E and LS1 is also shorter than that between E and LS2. In supercoiled DNA, the spatial separation of two sites is largely independent of their contour separation so E will sometimes be closer to LS2 than LS1. The lower pictures show catenanes containing one large and one small ring of DNA, with the larger ring in either its relaxed or supercoiled configurations. In the relaxed form, the small ring is free to move around the large ring in both rotational and lateral senses. In the supercoiled form, the small ring is located mainly at an apex of the superhelix and is largely blocked from lateral motion around the large ring.

Fig. 4. Relaxed DNA. The reactions contained two DNA substrates, both at 25 nM, and 0.2 nM EcoRV in reaction buffer at 37°C. Samples were removed at various times after the addition of EcoRV and analysed as in Materials and methods for the extents of cleavage of both DNA. Data from four repeats are shown: circles, squares, triangles and diamonds denote the individual experiments. The two substrates are: in (A), relaxed plasmid (filled data points) and minicircle (unfilled data points); in (B), relaxed catenane (filled points) and minicircle (unfilled points); and in (C), relaxed plasmid (filled points) and relaxed catenane (unfilled points). The plasmid and the catenane used here had been treated with topoisomerase I.

Fig. 5. ClaI on supercoiled and relaxed DNA. The reactions contained the plasmid pDG2 and the catenane (both at 25 nM) and ClaI (5 U/ml) in reaction buffer at 37°C. Samples were removed at various times and analysed as in Materials and methods for the extents of cleavage of both substrates. Data from two reactions are shown: circles and squares denote the individual experiments. The filled data points mark the cleavage of the plasmid and the unfilled points the catenane. In (A), both DNA contained their natural levels of supercoiling. In (B), both DNA had been relaxed with topoisomerase I.