Figure 3. Recombinant Human TDP2 Excises Denatured but Not Native TOP3Bcc.

(A) Oligonucleotide substrate (69-mer) with main TOP3B site (17 nucleotides from the 5′ end).

(B) Recombinant human TDP2 does not excise native TOP3Bccs. The oligonucleotide (300 nM) was incubated with recombinant human TOP3B (4 μM). TOP3Bcc formation results in a slower migrating band (lane 2) (for full gel and additional minor bands see Figure S3A). TOP3Bccs were incubated with 1 or 3 μM recombinant TDP1 (lanes 7 and 8) or TDP2 (lanes 5 and 6). Benzonase (3 and 9 units, lanes 3 and 4,) was used as a positive control to degrade the oligonucleotide and release TOP3B. Samples were immunoblotted with anti-TOP3B antibody after SDS-PAGE.

(C) Recombinant human TDP2 excises denatured cellular DNA and RNA TOP3Bccs. HEK293 cells were transfected with FLAG-tagged R338W-TOP3B, and protein-nucleic acid adducts were recovered by RADAR assay. After incubation with recombinant TDP2 (3 and 6 μM, lanes 4 and 5), H351A TDP2 (3 and 6 μM, lanes 6 and 7), D262A TDP2 (3 and 6 μM, lanes 8 and 9), benzonase (250 units, lane 2), or MNase (300 units, lane 3), released TOP3B was detected by immunoblotting with anti-FLAG antibody after SDS-PAGE. Loading was tested by slot blotting and probing with anti-dsDNA antibody.

(D) Recombinant human TDP2 excises cellular RNA TOP3Bccs. HEK293 cells were transfected with R338W-TOP3B. Covalent protein-RNA adducts were isolated using TRIzol. After incubation with recombinant TDP2 (3 and 6 μM, lanes 4 and 5), H351A TDP2 (3 and 6 μM, lanes 6 and 7), D262A TDP2 (3 and 6 μM, lanes 8 and 9), benzonase (250 units, lane 2), or an excess amount of RNase A (200 μg/mL) and RNase T1 (200 units/ml) mix (lane 3), released TOP3B was detected by immunoblotting with anti-FLAG antibody after SDS-PAGE. Loading was tested by methylene blue staining (RNA).

See also Figure S3.