Fig. 3: TOP1-PARylation enhances TDP1 recruitment without enhancing TOP1cc hydrolysis.

a TOP1-TDP1 interaction was enhanced by PARGi and inhibited by PARPi. Following transfection of 6×His-tagged TOP1 expression construct, HEK293 cells were treated with the indicated drugs before His-tag pulldown using Ni-NTA agarose under native conditions. The pulldown and input samples were Western blotted with the indicated antibodies. b Proximity Ligase Assay (PLA) confirming that inhibiting PARG enhanced TOP1-TDP1 interactions. U2OS cells were treated with CPT (20 µM, 1 h), PARGi (10 µM, 2 h), or CPT + PARGi (pre-treatment with PARGi for 1 h then co-treatment with CPT and PARGi for 1 h), followed by PLA assay. The scale bar represents 3 μm. c ICE assay showing that TDP1 failed to resolve hyper-PARylated TOP1-DPCs in vivo. HEK293 cells transfected with empty vector (EV) or TDP1-FLAG overexpression plasmid were treated with CPT (2 µM) for 1 h in the absence or presence of PARGi or BTZ. Cells were collected for ICE assay. 2 µg of digested DNA from each ICE assay sample was subjected for slot-blotting using α-TOP1 antibody or α-dsDNA antibody. d Scheme for [³²P]-labeled TOP1cc preparation, PARylation, and TDP1 digestion assays. e Schematic representation of the reaction products generated by TDP1 with the DNA suicidal substrate alone, unmodified TOP1ccs, and PARylated TOP1ccs. f TDP1 acted on unmodified and PARylated TOP1-DPCs at high concentrations in vitro. Representative TDP1 assay as described in panel (d) showing the 3′-nucleosidase activity of TDP1 (left set of eight samples) and TDP1’s phosphodiesterase activity toward unmodified TOP1-DPC (middle 8 samples), which is facilitated by heat denaturation of TOP1cc but does not exhibit significantly altered  activity toward PARylated-TOP1cc (right eight samples). TDP1 concentrations: 0, 1.25, 25 and 500 nM.