Repair of linearized plasmids in cell extract. A) Preparation of Asf1-depleted cell extract. Depletion of Asf1A and Asf1B with respective siRNAs was monitored by western blot. B) Ligation of cohesive (EcoRI ends) and role of Asf1. The reaction was assembled as described in [15, 3]. Plasmid cut with EcoRI was incubated with Asf1-depleted extract or control, to monitor end-joining repair coupled with nucleosome assembly. The positions of the linearized and closed/relaxed forms of the plasmid are marked. C) Ligation of EcoRI/EcoRV-cut plasmid. The reactions contained 5 μCi [α-32P]dATP (0.1 mM final concentration) to label the plasmid by endogenous polymerases. In the left and right panels (autorad and EtBr), the stimulation of plasmid religation/supercoiling by added TLK1B is shown. The position of the linearized and ligated/supercoiled form is shown. Quantitation of the autorad in pixels (supercoiled form) is shown below each lane. D) Labeling by fill-in in Ku-depleted extract. The effect of TLK1B on end-labeling is shown in the absence of ligation. Quantitation of the autorad in pixels is shown below each lane.