Fig 4. “In gel one pot assay” analysis of the effect of FACT on the DNA accessibility towards HaeIII along the length of nucleosomal DNA in control and RSC treated nucleosomes.

(A, B) Effect of FACT on RSC-induced remosomes generation. (B) Preparative PAGE. Centrally positioned nucleosomes were treated with increasing amount of FACT in the presence of 0.2 units of RSC and after arresting the reaction they were separated on native PAGE; last lane, nucleosomes treated with 5-fold higher amount (1 unit) of RSC, in the absence of FACT; the first three lanes, untreated, and treated with FACT and with 0.2 units of RSC nucleosomes, respectively. The indicated bands (from 1 to 9) were excised from the gel and in-gel digested with 8 units of HaeIII for 10 minutes at 30°C. The cleaved DNA was then isolated and separated in 8% PAGE under denaturing conditions (B). The positions at the cleavage of the different dyads are indicated on the left; the numbers of each lane refers to the respective excised bands from the preparative PAGE (see A); ctrl, control, non-digested DNA; DNA, naked DNA used for reconstitutions of the nucleosomes digested with HaeIII. (C) Quantification of the data presented in (B).