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. 2000 Jan 15;28(2):570–581. doi: 10.1093/nar/28.2.570

Figure 7.

Figure 7

Figure 7

The proline-rich region stimulates DNA binding and DNA looping by hand Z2. (A) Gel mobility shift assays were used to assess the effect of the proline-rich region of DNA binding by GST-Z2. Equivalent amounts of GST-Z2 and GST-Z2ΔP were incubated with 32P-labeled DSR probe, the products were resolved by non-denaturing electrophoresis, and binding was visualized by autoradiography. GST-Z2 forms at least four gel shift complexes with the DSR (A–D) whereas GST-Z2ΔP forms a single complex. Competition assays showed binding by GST-Z2ΔP was specific for the DSR (data not shown). (B) GST-Z2 and GST-Z2ΔP were tested for their ability to mediate DNA looping using a ligation enhancement assay. In this instance ligation of the linear pCH127 control was allowed to proceed for 20 min in order to visualize a more complex array of ligation products. Lanes 6–8 and 9–11 contained 5, 50 or 500 ng of the indicated protein, respectively. (C) DNA looping between the USR and DSR by GST-Z2. The arrows indicate free ends of pCH127 that protrude from the loop complex. (D) The PRR is required for DNA looping. When incubated with GST-Z2ΔP looping between the USR and DSR in linear pCH127 was not observed by AFM.