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. 2024 Aug 8;13:e80687. doi: 10.7554/eLife.80687

Figure 2. SMC-5 represses intersister crossovers.

(A) Bar plot displaying the percent of crossover recombinant progeny identified in wild type and smc-5 ICR assays out of all recombinant progeny scored within individual 12 hr timepoint periods. Frequencies of recombinants identified overall in ICR assays is displayed in Figure 2—figure supplement 1. Wild type ICR assay data is shared in Figures 1A, 2A, B, 4A, Figure 1—figure supplement 2, Figure 2—figure supplement 1, and Figure 4—figure supplement 1. The total number of progeny scored in each dataset are (10–22 hr/22–34 hr/34–46 hr/46–58 hr timepoints) wild type: 28/25/22/15, smc-5(ok2421) 18/37/21/8. Specific progeny counts separated by experimental replicate are presented in Figure 2—source data 1. (B) Bar plot displaying the percent of crossover recombinant progeny identified in wild type and smc-5 ICR assays out of all recombinant progeny scored within the interhomolog window (22–58 hr post heat shock) and non-interhomolog window (10–22 hr post heat shock). Data is shared with panel (A). (C) Images of wild type and smc-5(ok2421) mutant bivalent chromosomes displaying an absence or presence of SCEs. Scale bars represent 1 μm. Dashed bordered insets contain cartoon depictions of the SCE and non-SCE bivalents that are outlined with dashed lines in the images to aid in visualizing exchange events. (D) Frequency of SCEs identified among wild type (n=49) or smc-5(ok2421) mutant (n=14) chromatid pairs scored. (E) Table displaying the percent of sequenced non-Unc progeny in wild type and smc-5 IH assays that showed signatures of mutagenic repair. Numbers in parentheses indicate the number of mutant worms out of the total number of sequenced progeny. Colored boxes indicate timepoints in which mutant progeny were identified. The overall frequency of interhomolog assay non-Unc progeny is displayed in Figure 2—figure supplement 2. Note that only mutations that allow for translation of functional UNC-5 protein can be detected (see Methods). In all panels, error bars represent 95% Binomial confidence intervals, dashed vertical lines delineate between timepoints within the interhomolog window (22–58 hr post heat shock) and non-interhomolog window (10–22 hr post heat shock), and p values were calculated using Fisher’s Exact Test.

Figure 2—source data 1. The source data for Figure 2A is provided.
The total number of ICR assay progeny with GFP+ or non-GFP+ phenotypes are listed. Wild type data is shared with Figure 1, Figure 1—figure supplement 2, Figure 2, Figure 2—figure supplement 1, Figure 4, and Figure 4—figure supplement 1.
Figure 2—source data 2. The source data for Figure 2D is provided.
The number of scorable chromatid pairs with SCE or no SCE events are listed for each image assessed in generating this dataset. Wild type data is shared with Figure 1C.
Figure 2—source data 3. The source data for Figure 2E is provided.
The total number of IH assay nonrecombinant, recombinant, mutant, or undetermined non-Unc progeny are listed. Wild type data is shared with Figure 1, Figure 1—figure supplement 3, and Figure 2—figure supplement 2.

Figure 2.

Figure 2—figure supplement 1. Intersister/intrachromatid repair (ICR) assay GFP+ progeny are elevated in smc-5 mutants.

Figure 2—figure supplement 1.

Stacked bar plots displaying the percent of all progeny scored in wild type and smc-5(ok2421) (A) or smc-6(ok3294) (B) ICR assays that were determined to be GFP+ noncrossover or crossover recombinants. Error bars represent the 95% Binomial confidence intervals for the frequencies of GFP+ progeny. Wild type ICR assay data is shared in Figures 1A, 2A, B, 4A, Figure 1—figure supplement 2, Figure 2—figure supplement 1, and Figure 4—figure supplement 1. smc-5 ICR assay data is shared in Figure 2 and Figure 2—figure supplement 1. The total number of progeny scored in each dataset are (10–22 hr/22–34 hr/34–46 hr/46–58 hr timepoints) wild type: 3921/3359/3664/2383, smc-5(ok2421) 1350/1771/1450/449, smc-6(ok3294) (10–22 hr/22–46 hr) 1855/2129. Specific progeny counts separated by experimental replicate are presented in Figure 2—source data 1. p values were calculated by Fisher’s Exact test comparing the sum totals of GFP+ progeny between groups. Vertical dashed lines demarcate the interhomolog window (≥22 hr post heat shock) and non-interhomolog window (10–22 hr post heat shock) timepoints.
Figure 2—figure supplement 2. Interhomolog repair is largely unperturbed in smc-5 mutants.

Figure 2—figure supplement 2.

(A) Stacked bar plots displaying the percent of all progeny scored in wild type and smc-5 IH assays that were determined to be noncrossover recombinants, crossover recombinants, non-Unc mutants, or undetermined non-Unc. (B) Percent of all recombinant progeny identified within the interhomolog window of wild type and smc-5 IH assays that were crossover recombinants. Error bars represent the 95% Binomial confidence intervals for the frequencies of non-Unc progeny. Wild type data is shared between Figures 1E and 2E, Figure 1—figure supplement 3, and Figure 2—figure supplement 2. The total number of progeny scored in each dataset are (10–22 hr/22–34 hr/34–46 hr/46–58 hr timepoints) wild type: 1308/1695/1592/562, smc-5 1031/1466/1369/596. Specific progeny counts separated by experimental replicate are presented in Figure 2—source data 3. p values were calculated by Fisher’s Exact Test comparing the overall frequency of non-Unc progeny (A) or proportion of crossover recombinants of all confirmed recombinant progeny (B, see Materials and methods). Vertical dashed lines demarcate the interhomolog window (22–58 hr post heat shock) and non-interhomolog window (10–22 hr post heat shock) timepoints.
Figure 2—figure supplement 3. Illustrations of mutants identified in smc-5 IH assays.

Figure 2—figure supplement 3.

Illustrations of unc-5 lesions identified in IH assay non-Unc progeny in smc-5 mutants (Figure 2, Figure 2 - figure supplement 2). Specific mutation signatures are numbered and are separated by horizontal dashed grey lines. The wild type unc-5 locus sequence at the site of Mos1 excision and the DSB product generated by Mos1 excision are displayed on the top (Numbered 1). Blue letters indicate a duplicated TA at the site of Mos1 insertion in the unc-5(ox171) locus, while yellow letters indicate the 3 nt 3’ overhangs left following Mos1 excision (Robert et al., 2008). Purple letters with bars indicate complementary bases flanking the deletion site. Red letters struck through with red lines indicate bases in the damaged locus which were deleted to produce the final product.