Fig 1: E152Q mutation that ablates Mg2+–TDP2 binding also abolishes 3′-TDP activity in vitro and in vivo.A, E152Q mutation abolishes the 3′-TDP activity of the purified TDP2 protein. WT or E152Q TDP2 (0, 17.3, 34.6, 69.2, 138.4, 276.8, and 553.6 nM) were incubated with the 3′-YP substrate in a TDP reaction buffer containing 1 mM Mg2+ at 37 °C for 10 min, and the products were analyzed using denaturing PAGE (upper). Conversion (%) of 3′-YP to the product (3′-P) is plotted against the TDP2 concentration (lower). B, survival curves of WT and TDP1/TDP2-mutated TK6 cells following CPT treatment. Survival rates of WT, TDP1−/−, TDP2−/−, TDP2E152Q/E152Q, TDP1−/−/TDP2−/−, and TDP1−/−/TDP2E152Q/E152Q (clones #1 and #2) cells were measured using colony-forming assays (as described for ETP treatment in Fig. 2G). C, model of TOP1cc repair. In the first step, the irreversibly trapped TOP1cc is polyubiquitinated and proteolyzed to the peptide by the proteasome. In the second step, the phosphotyrosyl bond linking DNA and the TOP1-derived peptide is hydrolyzed by TDP1 or TDP2, resulting in a 3′-phosphate end. Polynucleotide kinase/phosphatase (PNKP) removes the 3′-phosphate to generate a 3′-OH end and phosphorylates the 5′-OH end to produce a 5′-phosphate. 3′-OH and 5′-phosphate ends are ligated by LIGIIIα in the presence of XRCC1, that is, the gap-filling repair step. D, tail moments (raw data) of CPT-treated and untreated cells in alkaline comet assays. WT, TDP1−/−, TDP2−/−, TDP2E152Q/E152Q, TDP1−/−/TDP2−/−, and TDP1−/−/TDP2E152Q/E152Q (#1) cells were treated with 25 μM CPT for 60 min or left untreated, and the tail moments were measured without a postincubation step using alkaline comet assays. In total, 50 cells were analyzed for each sample, and experiments were conducted in triplicate for each cell type. Tail moments of individual cells (an arbitrary number of SSBs) of each cell type from three experiments are plotted vertically in three separate columns. Significant differences were determined using a Wilcoxon rank sum test: ∗∗p < 0.01. E, repair kinetics of SSBs in WT and TDP1/TDP2-mutated TK6 cells. Indicated cells were exposed to 25 μM CPT for 60 min and then incubated in CPT-free culture media for 30 or 60 min. Tail moments were measured using alkaline comet assays (as in panel D). Tail moments were standardized to those at 0 min of repair (bars on CPT). Percentages of remaining SSBs after 30 and 60 min were calculated relative to those at 0 min and are presented as the fractions of SSBs remaining. Data are means ± SDs from three biological replicates. Significant differences were identified using Student’s t test: ∗∗p < 0.01. Typical alkaline comet images and the raw data of tail moments before standardization are shown in Fig. S7, A and B, respectively. F and G, repair kinetics of CPT-induced TOP1ccs in WT and TDP1/TDP2-mutated TK6 cells. Cells were treated with CPT (as in panel D), and genomic DNA was isolated after the indicated postrepair incubation times. DNA was slot-blotted on a nitrocellulose membrane, and the membrane was probed with anti-TOP1 (F) or anti-TOP1cc (G) antibodies. Quantities of TOP1 (F) and TOP1-derived peptides (G) covalently linked to DNA were standardized to those after 0 min of repair. Remaining damage is presented for each cell type (as in panel E; also see panel E for the bar colors). Significant differences were identified using Student’s t test: ∗∗p < 0.01, ∗p < 0.05, and n.s. = not significant. CPT, camptothecin; ETP, etoposide; SSB, single-strand break; TDP, tyrosyl-DNA phosphodiesterase; TOP, topoisomerase; TOP1cc, TOP1 cleavage complex.
Fig 2: Sensitivity of DNA repair–deficient cells to CPT, ETP, and CTNAs that produce 3′-blocking DNA lesions.A, CPT and ETP sensitivity profiles of selected DNA repair–deficient TK6 cells. Sensitivity of the mutant relative to the WT was determined as described in the Experimental procedures. Negative and positive scores indicate the sensitivity and resistance of a given cell line to the drug, respectively. Relative sensitivity was calculated as follows: log2 [(LD10 in mutant cells)/(LD10 in WT cells)]. Each bar is colored according to the DNA repair function category: red, TDP-related repair; blue, DSB repair; yellow, base excision repair (BER); gray, nucleotide excision repair (NER); black, DNA–protein crosslinks repair; purple, postreplication repair; and green, mismatch repair (MMR). Error bars are SDs of the mean of three independent assays. B–E, sensitivity of TDP1−/−TDP2E152Q/E152Q cells to CTNAs that produce 3′-blocking DNA lesions. Indicated cells were treated with ABC (B), AZT (C), Ara-C (D), or gemcitabine (E). Survival rates were measured using colony-forming assays (as shown in Fig. 2G). ABC, abacavir; AZT, 3′-azido-3′-deoxythymidine; CPT, camptothecin; CTNA, chain-terminating nucleoside analog; DSB, double-strand break; ETP, etoposide; TDP, tyrosyl-DNA phosphodiesterase.
Fig 3: Common divalent metal ion requirements of the 3′- and 5′-TDP activities of TDP2.A, 5′-TDP activity of TDP2. Top: Schematic representation of the 5′-TDP activity assay. 5′-TDP activity catalyzes the hydrolysis of the 5′-phosphotyrosyl bond and converts the duplex 5′-YP substrate (19-mer) to a product with a 5′-phosphate end (5′-P; 19-mer). Middle: Representative gels showing the results of 5′-TDP activity assays. The duplex 5′-YP substrate was incubated with TDP2 (0, 1, 2, 4, 8, 16, and 32 nM) or TDP1 (0, 10, 20, 40, 80, 160, and 320 nM) in a TDP reaction buffer containing 1 mM MgCl2 in the absence (−) or presence (+) of 50 mM EDTA at 37 °C for 10 min. Products were separated via denaturing PAGE and detected using autoradiography. Positions of the 32P-radiolabeled substrate (5′-YP) and the product (5′-P) are indicated. A standard marker for 5′-P (separately prepared) was also separated in the rightmost lanes of the gels. Bottom: Conversion (%) of the 5′-YP substrate to the product (5′-P) at different TDP1 and TDP2 concentrations in the absence (−) or presence (+) of EDTA. B, 3′-TDP activity of TDP2. Top: Schematic representation of the 3′-TDP activity assay. 3′-TDP activity catalyzes the hydrolysis of the 3′-phosphotyrosyl bond and converts the single-stranded 3′-YP substrate (18-mer) to a product with a 3′-phosphate end (3′-P; 18-mer). Middle: Representative gels showing the results of 3′-TDP activity assays. The single-stranded 3′-YP substrate was incubated with TDP1 (0, 1.25, 2.5, 5, 10, 20, 40, and 80 nM) or TDP2 (0, 17.3, 34.6, 69.2, 138.4, 276.8, and 553.6 nM) in a TDP reaction buffer containing 1 mM MgCl2 at 37 °C for 10 min. Products were separated via denaturing PAGE. Positions of the 32P-radiolabeled substrate (3′-YP) and the product (3′-P) are indicated. A standard marker for 3′-P (separately prepared) was also separated in the rightmost lanes of the gels. Bottom: Conversion (%) of the 3′-YP substrate to the product (3′-P) at different TDP1 and TDP2 concentrations. The broken line indicates 50% conversion. C, effect of Mg2+ concentration on the 3′-TDP activity of TDP2. Left: The 3′-YP substrate was incubated with 553.6 nM TDP2 (upper) or 80 nM TDP1 (lower) in a TDP reaction buffer containing MgCl2 (0.0001, 0.001, 0.01, 0.1, and 1 mM) in the absence (−) or presence (+) of 50 mM EDTA, and the products were separated via denaturing PAGE. The rightmost lane of the gels indicates the standard marker for the product (3′-P). Right: Conversion (%) of the 3′-YP substrate to the product (3′-P) by TDP2 and TDP1 at different concentrations of Mg2+ in the absence (−) or presence (+) of EDTA. D, effects of different divalent metal ions on the 5′-TDP activity of TDP2. Upper: Duplex 5′-YP substrate was incubated with TDP2 (0, 1, 2, 4, 8, 16, and 32 nM) in a TDP reaction buffer containing 1 mM Mg2+, Mn2+, Ca2+, Co2+, or Zn2+, and the products were separated via denaturing PAGE. Lower: Conversion (%) of the 5′-YP substrate to the product (5′-P) by TDP2 in the presence of Mg2+, Mn2+, Ca2+, Co2+, or Zn2+. E, effects of different divalent metal ions on the 3′-TDP activity of TDP2. Upper: Single-stranded 3′-YP substrate was incubated with TDP2 (8, 16, 32, 64, 129, and 257 nM) in a TDP reaction buffer containing 1 mM Mg2+, Mn2+, Ca2+, Co2+, or Zn2+, and the products were separated via denaturing PAGE. Lower: Conversion (%) of the 3′-YP substrate to the product (3′-P) by TDP2 in the presence of Mg2+, Mn2+, Ca2+, Co2+, or Zn2+. The data in panels A–E are the means ± SDs of at least three independent experiments. TDP, tyrosyl-DNA phosphodiesterase.
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