Fig 2. The simultaneous presence of both FACT and RSC, but not FACT alone, is required for efficient UDG excision of uracil at nucleosomal DNA sites oriented towards the histone octamer.
Centrally positioned nucleosomes were reconstituted by using 32P 5’-labeled 255 bp 601 DNA fragment containing randomly incorporated uracil residues. Lanes 1–6: analysis of the UDG enzymatic activity within the nucleosomal DNA. The nucleosome solution was incubated with the indicated increasing (nine-fold step) amount of UDG for 60 minutes at 30°C and the cleavage pattern of the isolated DNA was analyzed using PAGE under denaturing conditions; lane 2: no UDG added; lanes 1, 25: •OH footprinting of the nucleosomes. Lanes 7–12: RSC induces a highly efficient UDG-mediated excision of uracil at inward facing sites within the nucleosome. Nucleosomes were incubated with increasing (two-fold step) amount of RSC (units) for 50 min at 30°C, and after arresting the reaction they were treated with 1.2x10-2 units of UDG and the isolated cleaved DNA analyzed on denaturing PAGE; lane 7, control with no RSC added in the reaction. Lanes 13–19: FACT facilitates the RSC-dependent UDG excision of uracil at inward facing sites within the nucleosome. Nucleosomes were incubated with increasing (2-fold step) amount of FACT in the presence of 0.2 units of RSC and, after arresting the reaction they were treated with 1.2x10-2 units of UDG. The cleaved purified DNA was analyzed on denaturing PAGE; lane 13, control containing 1.6 pmol of FACT with no RSC added. Note that the excision of uracil by UDG is unaffected at this highest concentration of FACT used in the experiment. Lanes 20–24: UDG cleavage pattern of the naked 255 bp 601 fragment. The experiment was carried out as described in Lines 2–6, but with nine-fold smaller concentration of UDG on each respective point; on the left is shown schematics of the nucleosome.