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. 2022 May 27;50(10):5652–5671. doi: 10.1093/nar/gkac408

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© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 1. — t op-3-ZnF is a hypomorph allele. (A) Schematic representation of the top-3 gene, depicting the conserved topoisomerase domain (light blue) and the GRF zinc finger (ZnF) motif (green). The jf153 (top-3-ZnF) allele encodes a truncated protein that lacks the ZnF motif. (B) Western blot analysis of TOP-3::HA and TOP-3-ZnF::HA in whole worm extracts. wild-type (wt) worms were used to control for the specificity of the antibody. The TOP-3::HA protein is 100 KDa in size and the TOP-3-ZnF::HA protein lacks 45 amino acids. Actin was used as the loading control. Right panel: western blot quantification of three biological replicates per sample. Error bars indicate the mean and SD. (C) Hatch rates for the indicated genotypes: wt, 99.8% ± 0.3%, n = 18; top-3-ZnF, 98.8% ± 0.7%, n = 17; rtel-1(tm1866), 84.7% ± 34.3%, n = 8; rtel-1(tm1866); top-3-ZnF, 60.1% ± 43.4%, n = 9; mus-81(tm1937), 95.0% ± 3.0%, n = 8; mus-81(tm1937); top-3-ZnF, 70.1% ± 32.5%, n = 7. n = number of hermaphrodites. The histogram indicates the mean ± (SD). * P = 0.02, *** P = 0.0003, **** P < 0.0001, as determined using the Mann–Whitney test; non-significant differences are not shown. (D) The percentage of RAD-51 foci classes in each of the seven zones of the gonad in the indicated genotypes. For each zone, the average number of RAD-51 foci/nucleus was calculated from three gonads per genotype. Bottom right panel: schematic representation of the C. elegans gonad divided into seven equal zones. (E) Quantification of DAPI bodies in the −1 diakinesis oocyte in the indicated genotypes. Mean ± SD and number (n) of DAPI bodies per genotype: wt, 6 ± 0, n = 50; top-3-ZnF, 5.96 ± 0.3, n = 154; spo-11(ok79), 12 ± 0, n = 50; spo-11(ok79); top-3-ZnF, 12 ± 0, n = 50; rtel-1(tm1866), 6.2 ± 0.4, n = 34; and rtel-1(tm1866); top-3-ZnF, 6.1 ± 0.3, n = 41. Right panel: qualitative analysis of the −1 diakinesis oocyte of the indicated genotypes. n = number of DAPI bodies and the percentage of the different types of DAPI bodies observed per genotype: mus-81(tm1937), n = 90, bivalents = 70.2%, dissociated bivalents = 8.5%, DNA fragments = 14.9%, univalents = 5.3%, n/d (not defined) = 1.1%; mus-81(tm1937); top-3-ZnF, n = 50, bivalents = 44.6%, dissociated bivalents = 16.2%, fragments = 12.2%, univalents = 6.8%, n/d (not defined) = 20.3%. Insets show representative images of DAPI-stained diakinesis nuclei of the indicated genotypes. For top-3-ZnF, the dashed light blue circles indicate the presence of bridges and a doughnut-shaped bivalent.