Fig 1: Generation and complementation of BLM−/− RPE-1 cells with RPA-binding mutants of BLM and RMI1.a Western blots showing that BLM expression is undetectable in three separate BLM knockout RPE-1 clones. b Schematic showing the complementation strategy for adding back WT BLM/RMI1 proteins or mutant ones lacking RPA-binding motifs to BLM−/− cells treated with siRMI1. c Western blots showing the levels of exogenous GFP and BLM/RMI1 proteins delivered by lentivirus in WT or BLM−/− cells treated with siCtrl or siRMI1 as indicated. d Airyscan high-resolution confocal microscopy image time-course, showing the kinetics of recruitment of γH2AX, RPA2, and WT/mutant GFP-BLM proteins to laser-induced DSBs in representative cells. Zoomed-in images of laser lines are from the 60-min time-point images. Scale bars, 5 μm.
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).
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