FIG. 7.

Analysis of sequence-dependent association of Mma10b with DNA by agarose (A and B) and polyacrylamide gel (C) EMSA. (A) Binding of Mma10b to linear DNA fragments with and without the binding motif. The DNA concentration was 15 nM. Protein-DNA complexes were resolved by electrophoresis on 1.2% agarose gels. From lane 0 to 5, the protein concentrations were 0, 1, 2, 3.75, 7.5, and 15 μM, respectively. The presence or absence of Mma10b binding sites was predicted by the Motif Locator program with a score cutoff of 9.0. (B) Binding of Mma10b to closed, circular supercoiled and linear forms of the plasmid pB9. The linear plasmid was generated after digestion with HindIII. Based upon the Motif Locator program with a score cutoff of 9.0, the DNA sequences contained four potential Mma10b binding sites. The DNA concentration was 2 nM. Protein-DNA complexes were resolved by electrophoresis on 0.7% agarose gels. From lane 0 to 5, the protein concentrations were 0, 1, 2, 3.75, 7.5, and 15 μM, respectively. (C) Binding of Mma10b to ³²P-labeled 58-bp dsDNA fragments. The DNA concentration was 20 nM. Protein-DNA complexes were resolved by electrophoresis on an 8% nondenaturing polyacrylamide gel. The gel was exposed to X-ray film. From lane 0 to 10, the protein concentrations were 0, 0.02, 0.04, 0.08, 0.16, 0.31, 0.63, 1.25, 2.5, 5, and 10 μM, respectively. The upper panel used a DNA fragment with the wild-type sequence (positions 1307685 to 1307742 in the chromosome, which included “binding site a” in Fig. 8). The lower panel used a fragment where key bases in the motif were changed as indicated.