Fig 2: Similar DMC1 count elevation in Zcwpw1-/- and Prdm9-/- mice, compared to wild-type.(A) Testis chromosome spreads from 9- to 10-week-old Zcwpw1+/+ and Zcwpw1-/- mice were immunostained for DMC1 and SYCP3. Late (pseudo)-Pachytene cells are shown. These images are representative of the data obtained for three mice per genotype. Scale bar: 10 µm. (B) The number of DMC1 foci in cells from the indicated stages of prophase I were counted; see Figure 4—source data 1 for number of cells per stage per mouse. p-values are from Welch’s two sided, two sample t-test. L: Leptotene, Z: Zygotene, P: Pachytene. n = 3 mice for Zcwpw1-/- and wild-type, n = 2 for Prdm9-/-. Raw data in Figure 4—source data 1.Figure 4—source data 1.Raw data for DMC1, RAD51 and RPA2 foci counts.Stages: L, Leptotene; Z, Zygotene; P, Pachytene. WT: wild-type. Mouse ID is consistent across Figure 3—source data 1 and 2 and Figure 4—source data 1.

Fig 3: RPA-binding is required for the role of the BTR complex in response to DNA replication stress.a Quantification of CldU/IdU tract length ratios in DNA fiber assays showing that RPA-binding by BLM and RMI1 is required for replication fork restart (n = 3, one representative experiment shown). At least 100 DNA fibers were analyzed per sample. Significance was determined using the two-sided Mann–Whitney U test. b Colony survival assays showing RPA-binding by BLM and RMI1 is required for HU resistance (n = 3, error bars denote SEM). Cells were treated with the indicated doses of HU for 24 h. c Immunofluorescence confocal microscopy images of the indicated proteins, demonstrating that the RPA-binding BLM mutant (ΔRPA) is defective in its ability to form foci that colocalize with RPA2 in response to replication stress. Cells were treated where indicated with 2 mM HU for 24 h. d Quantification of the number of WT and ΔRPA GFP-BLM foci in the experiment shown in c). Significance was determined using the two-sided unpaired t test (n = 3, error bars denote SEM).

Fig 4: Recombination defects in mice lacking MCM8.(A) Chromosome spreads of Mcm8m/m pachytene-like cells with normal γH2AX staining (no flares), γH2AX flares, or γH2AX patches are shown on the left. Insets show higher magnification views of flares. Graph quantifies γH2AX staining patterns. Bars are means with standard deviations indicated for three mice. (B) Chromosome spreads depicting time course of DMC1 staining during meiotic prophase. (C) Quantification of DMC1 foci across meiotic prophase. (D) Quantification of RAD51 foci across meiotic prophase. (E) Quantification of RPA2 foci across meiotic prophase. In dot plots, foci overlapping with SYCP3-positive axes were scored. Each point is a count from one cell and means ± standard deviations for three mice are shown in blue lines. Results of two-tailed Mann Whitney tests are shown: ns is non-significant (p > 0.05), * is p ≤ 0.05; ** is p ≤ 0.01, *** p ≤ 0.001, and **** is p ≤ 0.0001. Cells were staged based on SYCP3-staining patterns: leptotene (Lept.), early zygotene (E. Zyg.), mid zygotene (M. Zyg.), late zygotene (L. Zyg.), early pachytene (E. Pach.), late zygotene-like (L. Zyg-like), early pachytene-like (E. Pach-like).

Fig 5: MCM8 functions downstream of resection to regulate recombination intermediates.(A) Overlap of hotspots identified by DMC1 SSDS in adult mice of the indicated genotypes. B6 wild type refers to B6 strain-specific hotspots. (B) Metaplots of DMC1 SSDS averages around B6 wild-type hotspots in adult mice. Sequencing reads originating from forward (fwd) and reverse (rev) strands are shown separately. Strand-specific reads were averaged and normalized by setting the highest value of the resection peak to 1. (C) Metaplots of RPA2 SSDS averages around B6 wild-type hotspots in adult mice. Reads originating from forward (fwd) and reverse (rev) strands are shown separately. (D) Genome-average S1-seq at B6 wild-type hotspots in adult mice. Reads from the reverse strand were flipped and combined with forward strand reads, averaged, and normalized by setting the height of the resection peak to 1. (E) Chromosome spreads immunostained for SYCP3 and MSH5. Insets show higher-magnification views of foci on chromosome axes. (F) Quantification of MSH5 foci in late zygotene and early pachytene-like cells. (G) Chromosome spreads of early to mid pachytene-like cells immunostained for SYCP3 and MLH1. (H) Quantification of MLH1 foci in pachytene-like cells. In (F) and (H), foci overlapping with SYCP3-positive axes were scored. Each point is a count from an individual cell. Means ± standard deviations for three mice are depicted in blue lines. Results of two-tailed Mann Whitney tests are shown: ****, p ≤ 0.0001. Staging was done using SYCP3-staining patterns, where early pachytene stage cells were defined as those with fully synapsed autosomes along with extensive X-Y synapsis.