FIG. 4.
EMSA measuring AID binding to bubble substrates. (A) EMSA with GST (−) or GST-AID (+) on HS1bub7 and HS1ds demonstrating the dependence of complex formation on the presence of a bubble structure (left and middle gels). EMSA comparing GST-AID and His-AID binding levels on HS1bub7 (right gel). (B) Representative EMSA experiment on a hot-spot bubble (HS3bub7) substrate. EMSA reactions were carried out in the presence of increasing amounts of the bubble substrate over a range of 0.15 to 50 fmol and electrophoresed in native conditions. In order to obtain half-saturation (Kd) values of interaction, the fraction of shifted substrate was quantitated for each lane and a bound-versus-free plot was generated. Filled circles represent results from an EMSA with UV cross-linking (the gel is shown) while empty circles represent results from an EMSA without UV cross-linking (gel not shown). (C) The same experiment as shown in panel B, except that CS3bub7 was used as the substrate in the EMSA experiment. (D) EMSA measuring the complex half-life of GST-AID bound to HS1bub7. Binding reactions were set up using 20 fmol of HS1bub7 as substrate and incubated for 45 min to allow for complex formation. A 500-fold excess (10 pmol) of unlabeled HS1bub7 was then added to the binding mixture, followed by incubation for various lengths of time as indicated on the x axis from 0 to 120 min prior to UV cross-linking of the reaction. The start times of the EMSA reactions were staggered such that all time points ended concurrently. Each time point was done in duplicate starting with 0, 5, 10, 30, 60, and 120 min of incubation after the addition of unlabeled HS1bub7. The amount of bound substrate after each incubation time with cold competitor is expressed as a percentage of the total bound substrate at time zero (no incubation with competitor) and shown on the y axis.