In vitro complementation of Tdp1 deficiency indicates a stabilized enzyme-DNA adduct from tyrosyl but not glycolate lesions as a consequence of the SCAN1 mutation

Abstract

A homozygous H493R mutation in the active site of tyrosyl-DNA phosphodiesterase (TDP1) has been implicated in hereditary spinocerebellar ataxia with axonal neuropathy (SCAN1), an autosomal recessive neurodegenerative disease. However, it is uncertain how the H493R mutation elicits the specific pathologies of SCAN1. To address this question, and to further elucidate the role of TDP1 in repair of DNA end modifications and general physiology, we generated a Tdp1 knockout mouse and carried out detailed behavioral analyses as well as characterization of repair deficiencies in extracts of embryo fibroblasts from these animals. While Tdp1(-/-) mice appear phenotypically normal, extracts from Tdp1(-/-) fibroblasts exhibited deficiencies in processing 3'-phosphotyrosyl single-strand breaks and 3'-phosphoglycolate double-strand breaks (DSBs), but not 3'-phosphoglycolate single-strand breaks. Supplementing Tdp1(-/-) extracts with H493R TDP1 partially restored processing of 3'-phosphotyrosyl single-strand breaks, but with evidence of persistent covalent adducts between TDP1 and DNA, consistent with a proposed intermediate-stabilization effect of the SCAN1 mutation. However, H493R TDP1 supplementation had no effect on phosphoglycolate (PG) termini on 3' overhangs of double-strand breaks; these remained completely unprocessed. Altogether, these results suggest that for 3'-phosphoglycolate overhang lesions, the SCAN1 mutation confers loss of function, while for 3'-phosphotyrosyl lesions, the mutation uniquely stabilizes a reaction intermediate.

Figure 1. Generation of Tdp1^−/− mice

Tdp1 targeting strategy. (i) Structure of the mouse Tdp1 gene, exons 5–12. (ii) Targeting vector for generation of both conditional and total knockout of the Tdp1 gene. (iii) Structure of the initial targeted Tdp1 allele prior to Cre expression. Structure of the knockout (iv) or conditional knockout (v) allele of Tdp1 following transient expression of Cre in the targeted ES cells.

Figure 2. Behavior of Tdp1^−/− mice and deficiency in 3′ tyrosyl processing

(A) Motor activity and latency to fall from the rotarod was determined for male and female mice of each genotype and at each age: 3, 6, and 12 months in top, middle and bottom panels, respectively. Asterisk indicates that at the 3 month assessment, Tdp1^−/− female mice were significantly less active than Tdp1^+/+ mice, p < 0.05. (B) A radiolabeled (*) 3′-pTyr oligomeric substrate was treated with four-fold serial dilutions of brain tissue homogenates from a Tdp1^+/+, Tdp1^+/−, or Tdp1^−/− mouse for 1 h, subjected to denaturing gel electrophoresis, and phosphorimaged. The percent conversion from the tyrosyl substrate to its phosphate product was calculated by densitometry.

Figure 3. Fibroblast cell lines derived from embryos of each genotype possess corresponding capacities for 3′ tyrosyl processing and can be biochemically or genetically complemented

Whole cell extracts from mouse embryonic fibroblast cell lines derived from Tdp1^+/+ or Tdp1^−/− mouse embryos were incubated with a substrate mimicking a SSB with a 3′-pTyr end modification. Processed and unprocessed forms of the substrate are indicated. (A) Diagram of 3′-pTyr SSB substrate; asterisk represents the radioactive 5′ tag on the 3′-pTyr 18-mer. There is no gap between the tyrosyl modification and the adjoining 25-mer. The substrate was treated with (B) 40, 20, and 10 μg of whole cell extracts from Tdp1^+/+ and Tdp1^−/− cell lines, and 40, 20, 10, and 5 μg of whole cell extract from the complemented Tdp1^−/− cell line for 1 h. Lanes 7–9 contain reactions with 40 μg of Tdp1^−/− extract, and 5, 1, and 0.2 ng of affinity-purified FLAG-TDP1, respectively. Presence of Tdp1 was also determined in MEF lysates from all three Tdp1 genotypes, as well as MEFs stably complemented by LV-FLAGhTDP1-Red transduction (“−/− comp”), by western blotting with anti-hTDP1 antibody (C). The gel contains 15 and 30 μL of each MEF lysate, and an identical membrane was loaded and probed with anti-β-actin as a loading control. The SSB substrate was treated from 1 to 60 min with 35 μg Tdp1^−/− MEF extract, with (filled symbols) and without (open symbols) supplementation with 50 ng of purified wild-type His-TDP1. The percent conversion from the tyrosyl substrate to its hydroxyl intermediate and repair products was calculated by densitometry (D).

Figure 4. Tdp1^−/− fibroblasts are deficient in processing PG on DSBs

Plasmid substrates mimicking DSBs with either partially complementary 3′ overhang (A) or blunt (B) DNA ends bearing PG modifications were incubated with whole-cell extract (or boiled extract) from Tdp1^+/+, Tdp1^+/−, and Tdp1^−/− and Tdp1^comp MEF cell lines for 6 h. Reactions were deproteinized, nucleic acids were precipitated, digested with BstXI and TaqI, and subjected to denaturing gel electrophoresis (C and D). In (C), the overhang substrate was incubated with 2.5 or 5 μg extract per μL reaction volume. In (D), the blunt ended substrate was incubated with 5 μg (or 10 μg *) extract per uL reaction volume. In (D), rightmost lane contains radiolabeled marker of 35 bases. The blunt-end DSB substrate was also incubated for 15 min, 1 h, 3 h, or 6 h with 7 μg of Tdp1^+/+ or Tdp1^−/− cell extract per μL reaction volume, then deproteinized, precipitated, and digested (E). Rightmost lane contains radiolabeled marker of 11 bases. After a 6 h incubation of the 3′-PG blunt end DSB substrate with extract, the percent of PG remaining in the lane was calculated by densitometry (F). Graph on left includes data from 4 independent experiments, performed with multiple preparations of cell extract per genotype; error bars indicate standard error and *** indicates p<0.001. Rightmost graph shows quantification of (E), time course of PG processing with Tdp1^+/+ or Tdp1^−/− cell extract. The 6-h time point, conducted in triplicate, includes error bars that are contained within the symbols.

Figure 4. Tdp1^−/− fibroblasts are deficient in processing PG on DSBs

Plasmid substrates mimicking DSBs with either partially complementary 3′ overhang (A) or blunt (B) DNA ends bearing PG modifications were incubated with whole-cell extract (or boiled extract) from Tdp1^+/+, Tdp1^+/−, and Tdp1^−/− and Tdp1^comp MEF cell lines for 6 h. Reactions were deproteinized, nucleic acids were precipitated, digested with BstXI and TaqI, and subjected to denaturing gel electrophoresis (C and D). In (C), the overhang substrate was incubated with 2.5 or 5 μg extract per μL reaction volume. In (D), the blunt ended substrate was incubated with 5 μg (or 10 μg *) extract per uL reaction volume. In (D), rightmost lane contains radiolabeled marker of 35 bases. The blunt-end DSB substrate was also incubated for 15 min, 1 h, 3 h, or 6 h with 7 μg of Tdp1^+/+ or Tdp1^−/− cell extract per μL reaction volume, then deproteinized, precipitated, and digested (E). Rightmost lane contains radiolabeled marker of 11 bases. After a 6 h incubation of the 3′-PG blunt end DSB substrate with extract, the percent of PG remaining in the lane was calculated by densitometry (F). Graph on left includes data from 4 independent experiments, performed with multiple preparations of cell extract per genotype; error bars indicate standard error and *** indicates p<0.001. Rightmost graph shows quantification of (E), time course of PG processing with Tdp1^+/+ or Tdp1^−/− cell extract. The 6-h time point, conducted in triplicate, includes error bars that are contained within the symbols.

Figure 5. Complementation with either SCAN1 or H493N mutant Tdp1 fails to restore the wild-type phenotype for processing either pTyr or PG end modifications

The pTyr substrate mimicking a SSB depicted in Figure 3A was treated with 30 μg of whole-cell extract from Tdp1^+/+, Tdp1^−/− MEFs for 5, 10, and 20 min (A). The substrate was also treated with 30 μg of Tdp1^−/− MEF extract that had been supplemented with 50 ng of either wild type purified TDP1, the SCAN1 mutant form of TDP1, or the mutant H493N TDP1. Processed (OH), unprocessed (pTyr), and repaired forms of the substrate are indicated. The radioactivity remaining in the SCAN1 wells presumably represents an unresolved DNA-TDP1 adduct; bands running beneath the wells and above the repair bands may represent a proteolyzed form of the adduct (*). The substrate mimicking a DSB with partially complementary 3′ overhangs as depicted in Figure 4A was treated with 60 μg whole-cell extract from Tdp1^+/+, Tdp1^+/−, and Tdp1^−/− and Tdp1^comp MEFs for 6 h (B). The substrate was also treated with Tdp1^−/− MEF extract that had been supplemented with either 25 ng (1X) or 125 ng (5X) of either wild type purified TDP1, the SCAN1 mutant form of TDP1, or the mutant H493N TDP1.