Extended Data Fig. 5. Trapped PARP1 is modified in a RNF4-dependent manner.
a. Overexpression of RNF4-WT increased PARP1 ubiquitination under trapping conditions. HEK293 cells were transfected with Ubiquitin-Strep-HA in combination with either FLAG-RNF4-WT or M136S/R177A mutant (E2 binding mutant, dominant negative) expressing constructs. After treatment with MMS + Talazoparib, the cells were fractionated and ubiquitylated proteins were purified from the chromatin-bound fraction via Streptactin beads. Data shown represent 2 biological replicas. b. RNF4 depletion prevents PARP1 ubiquitination under PARP trapping conditions. Denaturing IP of UB-HA-STREP-expressing HEK293 cells, similar to Fig. 2b. Cells were depleted of RNF4 with either a 5’UTR sequence or Dharmarcon SMARTpool and were treated with 100 nM Talazoparib and 0.01% MMS. Pulldown was conducted with Streptactin beads. Data shown represent 2 biological replicas. c. In vitro SUMOylation assay as described in Fig. 3g. The reactions were incubated in the presence of SUMO1, which was subsequently detected by anti-SUMO1 antibody. The asterisk indicates the free SUMO1. Data shown represent 2 biological replicas.