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

Figure 1. BRC-1 represses intersister crossovers and error-prone repair.

(A) Bar plot displaying the percent of crossover recombinant progeny identified in wild type and brc-1 ICR assays out of all recombinant progeny scored. Frequencies of recombinants identified overall in ICR assays is displayed in Figure 1—figure supplement 2A. Wild type ICR assay data is shared in A and Figures 2A, B, 4A, Figure 1—figure supplement 2, Figure 2—figure supplement 1, and Figure 4—figure supplement 1. brc-1 ICR assay data is shared in A and Figure 4A, Figure 1—figure supplement 2, and Figure 4—figure supplement 1. The total number of recombinant 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, brc-1(xoe4) 24/43/36/9. Specific progeny counts separated by experimental replicate are presented in Figure 1—source data 1. (B) Images of wild type and brc-1(xoe4) mutant bivalent chromosomes displaying an absence or presence of sister chromatid exchanges (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. (C) Frequency of SCEs identified among wild type (n=49) or brc-1 mutant (n=26) chromatid pairs scored. (D–E) Tables displaying the percent of sequenced GFP+ progeny in wild type and brc-1 ICR assays (D) or non-Unc progeny IH assays (E) that showed signatures of mutagenic repair. Numbers in parentheses indicate the number of mutant worms out of the total number of sequenced progeny. Shaded boxes indicate timepoints in which mutant progeny were identified. The overall frequency of interhomolog assay non-Unc progeny is displayed in Figure 1—figure supplement 3. In panels (D) and (E), note that only mutations that allow for translation of functional GFP (panel D) or UNC-5 (panel E) 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 1—source data 1. The source data for Figure 1A 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 supplement 2, Figure 2, Figure 2—figure supplement 1, Figure 4, and Figure 4—figure supplement 1.
Figure 1—source data 2. The source data for Figure 1C is provided.
The number of scorable chromatid pairs with SCE or no SCE events (no_SCE) are listed for each image assessed in generating this dataset. Wild type data is shared with Figure 2.
Figure 1—source data 3. The source data for Figure 1E is provided.
The total number of IH assay progeny with recombinant or mutant nonUnc phenotypes or Unc nonrecombinant phenotypes are listed. Wild type data is shared with Figure 1—figure supplement 3, Figure 2, and Figure 2—figure supplement 2.

Figure 1.

Figure 1—figure supplement 1. Diagram of the Intersister/Intrachromatid and Interhomolog assays.

Figure 1—figure supplement 1.

Cartoon depictions of how noncrossover and crossover events can be generated in the Intersister/Intrachromatid Repair assay (A) (Toraason et al., 2021c) and Interhomolog assay (B) (Rosu et al., 2011). Panel A is adapted from Toraason et al., 2021c.
Figure 1—figure supplement 2. Intersister/intrachromatid repair (ICR) assay GFP+ progeny are elevated in brc-1 and brc-1;brd-1 mutants.

Figure 1—figure supplement 2.

Stacked bar plots displaying the percent of all progeny scored in wild type and brc-1(xoe4) (A), brc-1(tm1145);brd-1(dw1) (B), and msh-2(ok2410) (C) ICR assays that were determined to be GFP+ noncrossover recombinants, crossover recombinants, or mutants. 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. brc-1 ICR assay data is shared in Figures 1A and 4A, Figure 1—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: 3921/3359/3664/2383, brc-1(xoe4) 2042/2017/2005/467, (10–22 hr/22–46 hr) brc-1(tm1145);brd-1(dw1) 1562/2340, msh-2(ok2410) 460/711. Specific progeny counts separated by experimental replicate are presented in Figure 1—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 1—figure supplement 3. Interhomolog repair is largely unperturbed in brc-1 mutants.

Figure 1—figure supplement 3.

(A) Stacked bar plot displaying the percent of all progeny scored in wild type and brc-1 IH assays that were determined to be noncrossover recombinants, crossover recombinants, non-Unc mutants, or undetermined non-Unc. The vertical dashed line demarcates the interhomolog window (22–58 hr post heat shock) and non-interhomolog window (10–22 hr post heat shock) timepoints. (B) Percent of all recombinant progeny identified within the interhomolog window of wild type and brc-1 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, brc-1 758/823/468/97. Specific progeny counts separated by experimental replicate are presented in Figure 1—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).
Figure 1—figure supplement 4. Illustrations of mutants identified in brc-1 ICR and IH assays.

Figure 1—figure supplement 4.

(A) Illustrated depiction of ICR assay GFP+ mutant identified in a brc-1 mutant background (Figure 1D, Figure 1—figure supplement 2). The partial tandem duplication produced (bottom) can best be parsimoniously explained by two independent strand invasion and extension events on either end of the DSB. For simplicity, intersister recombination is depicted in this diagram. However, intrachromatid templates could also have been engaged to produce the final product. (B) Illustrations of unc-5 lesions identified in IH assay non-Unc progeny in brc-1 mutants (Figure 1E, Figure 1—figure supplement 3). Specific mutation signatures 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 of panel (B). 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). In the panels displaying mutations identified, purple letters with bars indicate complementary bases flanking the deletion site. Red letters struck through with red lines indicate bases in the damaged locus that were deleted to produce the final product. Green letters indicate sites of nucleotide substitution mutations.