SCAN1 mutant Tdp1 accumulates the enzyme--DNA intermediate and causes camptothecin hypersensitivity

Abstract

Tyrosyl-DNA phosphodiesterase (Tdp1) catalyzes the hydrolysis of the tyrosyl-3' phosphate linkage found in topoisomerase I-DNA covalent complexes. The inherited disorder, spinocerebellar ataxia with axonal neuropathy (SCAN1), is caused by a H493R mutation in Tdp1. Contrary to earlier proposals that this disease results from a loss-of-function mutation, we show here that this mutation reduces enzyme activity approximately 25-fold and importantly causes the accumulation of the Tdp1-DNA covalent reaction intermediate. Thus, the attempted repair of topoisomerase I-DNA complexes by Tdp1 unexpectedly generates a new protein-DNA complex with an apparent half-life of approximately 13 min that, in addition to the unrepaired topoisomerase I-DNA complex, may interfere with transcription and replication in human cells and contribute to the SCAN1 phenotype. The analysis of Tdp1 mutant cell lines derived from SCAN1 patients reveals that they are hypersensitive to the topoisomerase I-specific anticancer drug camptothecin (CPT), implicating Tdp1 in the repair of CPT-induced topoisomerase I damage in human cells. This finding suggests that inhibitors of Tdp1 could act synergistically with CPT in anticancer therapy.

Figure 1

Tdp1 activity and protein in SCAN1 cells. (A) Sequencing gel analysis of Tdp1 activity assays with cell extracts from SCAN1 cells and related cell lines. Serial dilutions (10-fold) of extracts derived from cells that are either wt (wt-2, wt-5), homozygous for the H493R mutation (mut-1, mut-3, mut-4), or heterozygous at this locus (het-6, het-7) were incubated with substrate 12-Y, a 12-mer DNA oligonucleotide with a 3′ tyrosine moiety. The product of the reaction is 12-P, a 12-mer with a 3′ phosphate. Substrate 12-Y alone was loaded in lane 1. (B) Tdp1 assays of five-fold serial dilutions of recombinant wt and H493R Tdp1 proteins using substrate 12-Y. Substrate alone is shown in lane 1. The gel shown is representative of two independent experiments. (C) Immunoblot analysis of whole-cell extracts of the seven cell lines shown in (A). Rabbit anti-human Tdp1 antiserum was used to detect Tdp1. Purified recombinant His-tagged wt Tdp1 was loaded as a control in the far-right lane. (D) Specific inhibition of Tdp1 activity by anti-Tdp1 antiserum. Substrate 20-Topep, a 20-mer single-stranded DNA oligonucleotide with a small topoisomerase I-derived peptide attached to the 3′ end, was used as the substrate (lane 1). The 20-P product of the Tdp1 reaction is shown in lane 2. In lanes 3–14, 10 μl of a 1:2 dilution of the indicated cell extract was tested for Tdp1 activity. Where indicated, the cell extracts were preincubated with anti-Tdp1 antiserum (lanes 7–10) or preimmune serum (lanes 11–14) prior to the addition of substrate. A typical experiment of more than three independent experiments is shown.

Figure 2

H493R mutant accumulates the covalent Tdp1–DNA reaction intermediate. (A) The indicated Tdp1 enzyme was incubated with substrate 20-Topep. The conditions for the wt enzyme shown in lanes 1 and 2 were the same as shown in Figure 3, lane 6, the conditions for the H493R mutant shown in lanes 3–5 were the same as shown in Figure 3, lane 10, and the conditions for the H493A mutant shown in lanes 6 and 7 were the same as shown in Figure 3, lane 14. Reactions were stopped with SDS and a portion of each was digested with trypsin as indicated. The product resulting from trypsin treatment of the Tdp1–DNA covalent intermediate is 20-Tdpep, and is predicted to consist of the 20-mer DNA with an 11-mer trypsin-resistant Tdp1 fragment attached to its 3′ end. The sample in lane 5 was treated with proteinase K after trypsin treatment, which results in a 20-mer with a smaller peptide (labeled with a star). (B) A subset of the reactions shown in (A) was analyzed by SDS–PAGE. Tdp1 proteins that are trapped in the reaction intermediate (20-Tdp) are radioactively labeled via the 5′ end-labeled 20-mer DNA. Lanes 1 and 2, lanes 3 and 4, and lanes 5 and 6 correspond to lanes 1 and 2, lanes 3 and 4, and lanes 6 and 7 of (A), respectively. The mobility of two molecular weight markers is indicated on the right side of the gel. All experiments shown were repeated several times with identical results.

Figure 3

Activity assays of wt and mutant forms of Tdp1. Five-fold serial dilutions of the indicated proteins (starting at 1.4 μM) were incubated with substrate 20-Topep for 10 min, treated with trypsin, and analyzed on a sequencing gel. 20-Tdpep denotes the covalent reaction intermediate after trypsin treatment and 20-P the product of the cleavage reaction. One representative experiment of three is shown here.

Figure 4

Half-life of the covalent Tdp1 H493R–DNA intermediate. (A) Tdp1 H493R and 20-Topep were incubated for 2 min (zero time point, lane 2) before samples were taken at the indicated time points (lanes 2–10). Samples were treated with trypsin and analyzed in a sequencing gel. Lane 1 contains the substrate 20-Topep alone. (B) The decay of the Tdp1–DNA covalent intermediate with time is plotted for the time-course samples shown in (A). Averages of three independent experiments are shown, with error bars representing standard deviations. Some error bars are too small to be seen.

Figure 5

CPT sensitivity of SCAN1 cell lines. (A) Two wt cell lines (wt-2, ▪; wt-5, ▴), two homozygous mutant SCAN1 cell lines (mut-3, □; mut-4, ▵), and one heterozygous cell line (het-6, ○) were grown in the presence of the indicated amounts of CPT and live cells were counted at 72 h. The percentage of live cells compared to the untreated control was determined as a function of the CPT concentration. This particular analysis was performed in triplicate and the results are shown with error bars representing the standard deviation. (B) The percentage of live cells is plotted versus time at 2.5 nM CPT for the experiment shown in (A).

Figure 6

Multiparameter flow cytometry analysis of cell survival, cell death, and early apoptosis. (A–C) Two wt cell lines (wt-2, ▪; wt-5, ▴) and two SCAN1 cell lines (mut-3, □; mut-4, ▵) were grown in the presence of the indicated amounts of CPT. The relative numbers of live, dead, and early apoptotic cells were determined at 48 h. Each data point shows the mean and standard deviation of triplicate samples. (A) The percentage of live treated cells as compared to live untreated cells at each data point is plotted as a function of the CPT concentration. (B) The difference between the percentage of dead cells in the treated cell population and the percentage of dead cells in the untreated control population is plotted as a function of CPT concentration. (C) The difference between the percentage of early apoptotic cells in the treated cell population and the percentage of apoptotic cells in the untreated control population is plotted as a function of CPT concentration.

Figure 7

Cell cycle distribution of live and early apoptotic wt and SCAN1 mutant cell lines. (A) Cells were either untreated or treated with 5 nM CPT for 24 h. The abscissas represent the DNA content per cell as measured by the fluorescence intensity of Hoechst 33342, and reflect the G1, S, and G2 states of the cell cycle as indicated. The ordinates represent cell numbers. (B) Analysis is the same as described for the 5 nM CPT treatment in (A), except that cell numbers indicate those cells with low SYTO11 staining reflective of early apoptosis. (C) Percentage of cells in S phase after a 24-h treatment with the indicated concentrations of CPT (average of three experiments).