Figure 7

CTCF exhibits orientation dependent directed bend at FII insulator element. (A) κ-DNA contains phased A rich tracts with an intrinsic bend toward the minor groove. FII insulator element points towards κ-DNA (forward) or away from it (reverse). Helical phasing between sites is varied by increasing length of DNA spacer (black box) by 2 bp increments (10 to 20 bp) over one helical turn of the DNA. Plasmids digested with RsaI and PvuII gave 391bp to 401bp probes containing FII site. NheI was included to cleave a plasmid backbone fragment. (marked with asterisk, does not bind CTCF). (B) Schematic diagram of cis and trans isomers. Cis isomer migrates more slowly than trans isomer. (C) Phasing experiment: FII site in forward orientation. Two CTCF DNA complexes are seen. Lower complex, formed by truncated CTCF, is obscured by vector backbone fragment (*, lane 5). Complex migrates most slowly when the protein-induced and sequence-directed bends are additive ("cis" isoform, lanes 1 and 6). When bends are out of phase ("trans" isoform), complex has greatest mobility (lanes 4 and 5). Relative mobilities of protein-DNA complexes normalized to those of free probes are plotted relative to spacer length. Best fit polynomial curve was determined using Microsoft Excel. (D) Phasing experiment: FII site in reverse orientation. Two CTCF DNA complexes are seen. Lower complex is obscured by contaminating radiolabeled vector fragment (7, 9 and 11). "Cis" isomer and "trans" isomers are formed in alternating manner when spacer length increases by 2 bp.