Fig. 3: Polygenic Brca2^Δ27/Δ27 + Rad51c^dah/dah mutations confer hyper-DNA replication and genome instability.

a Scatter plot of DNA fiber analysis of nascent IdU tract lengths incorporated before replication stalling with high concentrations of hydroxyurea (HU, 4 mM), measuring DNA fork protection. Data represent biologically independent samples from wild-type (n = 3), Brca2^Δ27/Δ27(n = 2), Rad51c^dah/dah (n = 3) and Brca2^Δ27/Δ27 + Rad51c^dah/dah (n = 2) measurement. In total, analysis was performed for wild-type (n = 293), Brca2^Δ27/Δ27(n = 399), Rad51c^dah/dah (n = 319) and Brca2^Δ27/Δ27 + Rad51c^dah/dah (n = 306) fibers. Brca2^Δ27/Δ27,Rad51c^dah/dah and Brca2^Δ27/Δ27 + Rad51c^dah/dah against WT: p < 0.0001. b Scatter plot of DNA fiber analysis of nascent CldU tract lengths incorporated with replication stalling with low concentrations of hydroxyurea (HU, 400 µM), measuring replication restart. Data represent biologically independent samples from wild-type (n = 4), Brca2^Δ27/Δ27(n = 3), Rad51c^dah/dah (n = 3), and Brca2^Δ27/Δ27 + Rad51c^dah/dah (n = 3) measurement. In total, analysis was performed for wild-type (n = 303), Brca2^Δ27/Δ27(n = 263), Rad51c^dah/dah (n = 282), and Brca2^Δ27/Δ27 + Rad51c^dah/dah (n = 495) fibers. Rad51c^dah/dah and Brca2^Δ27/Δ27 + Rad51c^dah/dah against WT: p < 0.0001. c Scatter plot of DNA fiber analysis of nascent IdU tract lengths incorporated before replication stalling with low concentrations of hydroxyurea (HU, 400 µM), measuring DNA fork protection at low stress conditions. Data represent biologically independent samples from wild-type (n = 4), Brca2^Δ27/Δ27(n = 3), Rad51c^dah/dah (n = 3) and Brca2^Δ27/Δ27 + Rad51c^dah/dah (n = 3) measurement. In total, analysis was performed for wild-type (n = 410), Brca2^Δ27/Δ27(n = 329), Rad51c^dah/dah (n = 337) and Brca2^Δ27/Δ27 + Rad51c^dah/dah (n = 658) fibers. Brca2^Δ27/Δ27 against WT: p = 0.0103, Rad51c^dah/dah against WT: p = 0.005 and Brca2^Δ27/Δ27 + Rad51c^dah/dah against WT: p < 0.0001. d Cellular sensitivity of bone marrow cells to varying concentrations of Mitomycin C (MMC) as indicated. The number of colonies of progenitor cells were determined after 14 days. Data represent biologically independent bone marrow samples from wild-type (n = 3), Brca2^Δ27/Δ27(n = 3), Rad51c^dah/dah (n = 3), and Brca2^Δ27/Δ27 + Rad51c^dah/dah (n = 4) mice. e Representative images of metaphase chromosome spreads in Brca2^Δ27/Δ27 + Rad51c^dah/dah MAFs. Similar structure designed as radial were observed in >100 independent samples. f Scatter dot blot of radial chromosome structures in metaphase spreads of mouse adult fibroblasts (MAF) after treatment with 2 or 20 ng/ml MMC as indicated. Data represent 3 independent experiments. In total, chromosome analysis was performed for 2 ng/ml MMC treated Brca2^Δ27/Δ27(n = 48) and Brca2^Δ27/Δ27 + Rad51c^dah/dah (n = 86) cells as well as for 20 ng/ml MMC treated Brca2^Δ27/Δ27(n = 56) and Brca2^Δ27/Δ27 + Rad51c^dah/dah (n = 48) cells. 2 ng/ml MMC-treated Brca2^Δ27/Δ27 + Rad51c^dah/dah against Brca2^Δ27/Δ27: p < 0.0001; 20 ng/ml MMC-treated Brca2^Δ27/Δ27 + Rad51c^dah/dah against Brca2^Δ27/Δ2: p = 0.0575; g Scatter dot blot of radial chromosome structures (20 ng/ml MMC) with or without transient expression of wild-type Rad51c. Data represent 2 independent experiments. In total, chromosome analysis was performed for GFP-transfected Brca2^Δ27/Δ27 + Rad51c^dah/dah (n = 28) cells and for Rad51c-transfected Brca2^Δ27/Δ27 + Rad51c^dah/dah (n = 41) cells. Brca2^Δ27/Δ27 + Rad51c^dah/dah against Brca2^Δ27/Δ27 + Rad51c^dah/dah + WT Rad51c p = 0.0608. h Scatter dot blot of spontaneous chromosome aberrations in metaphase spreads in MAFs. Data represent 3 independent experiments. In total, chromosome analysis was performed for untreated wild-type (n = 50), Brca2^Δ27/Δ27(n = 52), Rad51c^dah/dah (n = 46), and Brca2^Δ27/Δ27 + Rad51c^dah/dah (n = 53) cells. Brca2^Δ27/Δ27 + Rad51c^dah/dah against WT and Rad51c^dah/dah: p < 0.0001; Brca2^Δ27/Δ27 + Rad51c^dah/dah against Brca2^Δ27/Δ27: p = 0.002. i Representative images of metaphase chromosome spreads without exogenous damage. Similar pictures of metaphases with total number of aberrations for each genotype were observed in >10 independent samples. Error bars represent the standard error of the mean. Bars represent the mean of compiled data from biological repeats. p-values for DNA fiber analysis and the genomic instability analysis with or without transient expression of wild-type Rad51c (g) are derived using the two-tailed Mann–Whitney test, and the p-values for the genomic instability analysis (f, h) are derived using the one-way Anova test. *p < 0.1, **p < 0.01, ***p < 0.001, ****p < 0.0001.