(A) Schematic structure of zebrafish Tdp1 protein, depicting the nuclear localization signal (NLS), phosphodiesterase domain, and active site histidines H270 and H501 (64). Zebrafish H501 corresponds to human H493. (B) Alignment of Homo sapiens (hs) and D. rerio (dr) TDP1 protein sequence. Identical sequences are highlighted in black, and similar sequences are highlighted in gray. The sequence targeted for deletion and catalytic HKD motifs [HxK(x)₄D(x)₆GSxN] with catalytic sites (in red) are noted (64, 65). Yellow denotes amino acids that contact the DNA substrate during repair. Accession numbers are as follows: hsTDP1, ENST00000335725.8, and drTdp1, ENSDART00000150149.2. Sequences were aligned using the Protein BLAST website: https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins. (C) Whole-mount in situ hybridization for tdp1 mRNA in wild-type zebrafish at 24 hpf. Sense probes for tdp1 mRNA were used as a negative control.

(A) Sequence of oligonucleotide used for guide RNA (gRNA) synthesis. The scaffolding sequence is in purple; the target sequence is in green, and the T7 polymerase promoter is in blue. (B) Intron-exon structure of the D. rerio tdp1 gene. Exon 2 (in red) was targeted for mutation by Cas9; scale bar, 5000 bp. Photo credit: Ringaile Zaksauskaite, University of Sheffield (generated using http://wormweb.org/exonintron). (C) Sequences of the target region in tdp1^WT zebrafish and two isolated deletion alleles, tdp1^SH475 and tdp1^SH476. The 5-bp deletion (SH476; light blue box) and the 4-bp deletion (SH475; green square). (D) Tdp1^−/+ zebrafish were crossed and genotyped at adulthood; χ² = 2.941 with two degrees of freedom; two-tailed P value of 0.5316. (E) Diagram depicting the TDP1 activity assay showing a 5′ labeled oligomer with a 3′-phosphotyrosyl (PY) that is incubated with zebrafish protein lysate. Active TDP1 processes the 3′-PY into a phosphate group, resulting in a band shift on a DNA sequencing gel. (F) TDP1 activity assay was performed on 600 ng of lysate from 4-dpf embryos. (G) TDP1 activity assay was performed on fin clips from adult zebrafish. LB, lysis buffer control

(A to F) Adult zebrafish movement was recorded with a camera system for 6 hours. Time spent swimming at low (<30 mm/s) (A), medium (30 to 60 mm/s) (C), and high (E) speeds (60 mm/s or above) and count of times low (B), medium (D), and high (F) swimming speeds were plotted. Each of the 18 fish from either genotype was recorded twice (n = 36) and shown as average ± SEM. P values were calculated by two-tailed Student’s t test with Holm adjustment for multiple comparisons. (G to J) Four-dpf (G and H) and 5-dpf (I and J) embryos from a single female tdp1^−/− and male tdp1^−/+ incross were subjected to 3 cycles of 5-min darkness and 5-min light using the photomotor response assay. (G and I) Total distance traveled in each cycle was plotted in each data point as average ± SEM. P value, two-tailed Student’s t test. (H and J) Total distance traveled in all light or dark cycles was measured. Lines indicate average ± SEM. (I) Total distance traveled each cycle was plotted as average ± SEM. P values were calculated using two-tailed Student’s t test. DMSO, dimethyl sulfoxide. *P < 0.05; **P < 0.01.

(A to P) Zebrafish (27-month-old) were intraperitoneally injected with TPT (22.5 mg/kg) or DMSO (22.5 mg/kg) on two consecutive days for a final concentration of 45 mg/kg and monitored for 1.5 hours using a camera system 24 and 48 hours after the second injection. P values, two-tailed Student’s t test with Holm post hoc analysis for multiple comparisons. Total distance traveled (A and B), average speed (C and D), low speed count (E and F), low speed duration (G and H), medium speed count (I and J), medium speed duration (K and L), high speed count (M and N), and high speed duration (O and P) were quantified after the second injection. (Q) Tissues were dissected from 29-month-old fish and treated with 14 μM CPT for 2 hours and then examined for Top1-CC accumulation using CsCl fractionation and immunoblotting, as described in Materials and Methods. (R) Quantification of (Q); two biologically independent experiments for heart and gut and four for brain; for each independent repeat, either four hearts or four guts were pooled. One brain was used for each independent repeat; ±SEM. P values, two-tailed Student’s t test. n.s., not significant. *P < 0.05; **P < 0.01.

(A) Diagram of blind assay to assess CPT sensitivity showing embryos with severe body curvature or brain necrosis or lacking swim bladders. (B) Tdp1^−/+ fish were incrossed, and at 4 dpf, sibling embryos were treated with CPT (350, 500, and 750 nM) overnight. At 5 dpf, the most strongly affected embryos were blindly selected and genotyped. χ² = 4.902 with two degrees of freedom. The two-tailed P value is equal to 0.0862. (C) A female tdp1^−/− fish was crossed with a male tdp1^−/+ fish, and at 4 dpf, the embryos were treated with 500 and 1 μM CPT overnight. At 5 dpf, most strongly affected siblings were blindly selected at each concentration and genotyped. χ² = 0.391 with one degree of freedom. The two-tailed P value is equal to 0.5316. (D) Three-dpf embryos were treated with 14 μM CPT for 2 hours; then, TOP1-CC was examined using CsCl fractionation and immunoblotting, as described in Materials and Methods. (E) Quantification of (D); three biologically independent experiments, ±SEM. P values, two-tailed Student’s t test.

(A) Four-dpf zebrafish were treated with 500 nM CPT overnight, and lysates were incubated with a 3′ labeled oligonucleotide with a 5′-phosphotyrosyl (PY). TDP2 processes the phosphotyrosyl moiety into a phosphate group, resulting in a lower band on a DNA sequencing gel. We noted bands that are higher than the original substrate, which suggest further repair events taking place using the zebrafish lysate. Lysis buffer (LB) was used as a negative control and human embryonic kidney (HEK) 293 cell lysate as a positive control. (B to D) Four-dpf zebrafish embryos were treated with 500 nM CPT overnight, and total RNA was processed by reverse transcription qPCR (RT-qPCR). Transcript levels normalized to rps29 are shown; three biologically independent experiments, ±SEM. P values, two-way analysis of variance (ANOVA) with Holm post hoc analysis for multiple comparisons. (E) A model for the requirement of distinct DNA repair factors for Top1-CC repair during the zebrafish life span. We propose that during embryonic development, zebrafish use Apex2 and Ercc4 to repair Top1-induced DNA breaks. During adulthood, however, Tdp1 becomes essential for repairing Top1-CCs, and thus, tdp1^−/− fish develop a mild behavioral defect and CPT hypersensitivity in adulthood but not at embryonic stage. *P < 0.05; **P < 0.01; ***P < 0.001; and ****P < 0.0001.