Fig 3.

BPLF1 DUB activity disrupts polymerase η recruitment and focus formation. Polymerase η is shown as pol eta in the figure. (A) DUB activity decreases Polη recruitment to chromatin. 293T cells were transfected with BPLF1 1-246, exposed to UV or HU treatment, fractionated, and probed for Polη. The relative density indicates the percentage of Polη recruited to the chromatin. The bottom panels show expression of PCNA and ubiquitinated PCNA (PCNA-Ub) in the chromatin. (B) H1299 cells were transfected with YFP-tagged Polη and FLAG-tagged BPLF1 1-246 and BPLF1 C61S constructs and irradiated with UV. Cells were fixed, permeabilized, and incubated with fluorescent antibody against FLAG. Nuclei were detected with DAPI staining. (C) Foci from transfected immunofluorescence samples were counted and quantitated as the average number of foci per cell. The foci from 20 cells were counted. (D) Samples in which Polη foci formation was abolished. PCNA was also deubiquitinated by BPLF1 DUB activity. In samples transfected in tandem with those for immunofluorescence, H1299 cells were harvested after UV treatment and probed for PCNA by Western blotting. The bottom panel shows expression of BPLF1 1-246 and BPLF1 C61S.