Figure 7

Poly(ADP-ribose) polymerase 1 (PARP1) is polyubiquitinated by PHRF1. (a) Total cell extracts harvested from HA-PHRF1/HA-PHRF1^C108A-transfected HEK293T cells and siRNA-transfected HeLa cells were immunoblotted with indicated antibodies. (b) HA-PHRF1 and HA-PHRF1^C108A were co-transfected with FLAG-Ub or FLAG-Ub^K48R into HEK293T cells with the addition of MG132. Total cell extracts were immunoprecipitated (IP) with anti-PARP1 antibody and immunoblotted with anti-FLAG antibody to detect ubiquitinated PARP1. (c) NHEJ reporter H1299 cells were either transfected with HA-PARP1 or PARP1 short hairpin RNA (shRNA) for 24 h and then transfected with I-SceI expression plasmid for another 24 h. The proportion of enhanced green fluorescent protein (EGFP)-positive cells was determined by flow cytometry at 48 h after I-SceI transfection. Each experiment represented the mean±S.D. of three independent experiments. *P<0.05 and **P<0.01 compared with the control. (d) Ku70 were depleted by shRNA in alt-NHEJ reporter EJ2-GFP cells for 48 h and then co-transfected with I-SceI and HA-PHRF1 or empty HA vector for another 48 h. The proportion of GFP-positive cells was determined by flow cytometry. Each experiment represented the mean±S.D. of three independent experiments. *P<0.05 compared with the control. (e) A schematic model of PHRF1 in NHEJ repair. PHRF1 constitutively associates H3K36me2/3 via PHD domain and NBS1 by pSDpTE motif. When DSBs occur, PHRF1 may move to DSBs with the assistance of NBS1 to ubiquitinate PARP1 for NHEJ repair. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HA, hemagglutinin; Ub, ubiquitin; WB, western blot