Fig 2: Embryonic and maternal ERCC1 limit retrotransposition during development.a, Quantification of frequency of spliced eGFP copies at E18.5 in wild type, Fanca−/−, Fancd2−/−, Fancd2−/K559R or Ercc1−/− by ddPCR. Each point represents one embryo. b, The strategy to collect oocytes from either Ercc1+/− or Ercc1f/f Zp3-Cre females. c, Expression of Ercc1 was quantified by single-cell qPCR in oocytes. Expression was normalized to Gapdh and made relative to Ercc1+/−. Each point represents one oocyte. d, Ercc1−/Δ embryos were assessed when born from Ercc1f/fZp3-Cre females and Ercc1+/− males. The frequencies at E18.5 (top) or E3.5 (bottom) were assessed. P values were calculated by a two-sided Fischer’s exact test. Exp., expected; Obs., observed. e, The frequency of Ercc1−/− embryos was assessed at E18.5 when born from Ercc1+/− female and Ercc1+/− male. P values were calculated by a two-sided Fischer’s exact test. f, Representative ddPCR plots showing the frequency of spliced eGFP and Hprt control in E18.5 embryos from Ercc1−/− or Ercc1−/Δ embryos from Ercc1+/− or Ercc1f/fZp3-Cre mothers, respectively. g, Quantification of frequency of spliced eGFP copies at E18.5 in wild-type Ercc1−/− or Ercc1−/Δ tissues from Ercc1+/− or Ercc1f/f Zp3-Cre mothers, respectively. Each point represents one embryo. Data represent mean and s.e.m. Unless otherwise specified, P values were calculated by a two-tailed Mann–Whitney U-test (NS, not significant P > 0.05, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001).Source data

Fig 3: Fluctuation analysis in K562 L1-G418R cell-lines, LINE-1 protein expression in mutant cell lines, and cell fitness in complemented human Fanconi fibroblasts.Independent cultures of each mutant were established from single cells and treated with DOX for 10 days. Each culture was then plated in one well of a 6-well plate containing semi-solid methylcellulose media and G418. Plating efficiency was determined for four cultures by performing three 10-fold serial dilutions and plating without G418. Statistical analysis was performed as described previously46. n = 3 independent experiments, the dot represents the mean and the error bars represent the s.e.m., P values were calculated by two-tailed Mann-Whitney U-test; a) SLX4, XPF and ERCC1 (SXE), b) Fanconi anemia (FA), c) Homologous recombination (HR), d) Translation synthesis (TLS), e) Nucleotide excision repair (NER), f) Non-homologous end joining (NHEJ) and g) Bloom syndrome helicase (BLM). h) Immunoblot for histone H3, LINE-1 ORF1p and LINE-1 ORF2p in wildtype, ΔFANCA, ΔFANCD2 and ΔXPF K562 L1-G418R cells after DOX treatment; H3 loading control (representative data from 2 independent experiments). i-k) P220 (ΔFANCA) and PD331 (ΔFANCC) human fibroblasts were complemented by ectopically expressing FANCA and FANCC cDNA, respectively. The cellular sensitivity of these cell lines to mitomycin C was tested and compared with the previously complemented cell line PD20 (ΔFANCD2); i) PD20 and PD20 complemented cell lines, j) PD220 and PD220 complemented cell lines, k) PD331 and PD331 complemented cell lines (data in i-k represent mean and s.e.m.; n = 3 independent experiments). Source data

Fig 4: Interstrand crosslink repair factors restrict LINE-1 retrotransposition and preserve genomic stability.a, Image of K562 L1-G418R wild-type and mutant colonies grown in semisold methylcellulose media following DOX treatment and G418 selection. b, L1 retrotransposition measurement using K562 L1-G418R wild-type and mutant cells (sg1 and sg2, two different sgRNAs). Each dot corresponds to an independent experiment. c, Representative western blot of FANCD2 following 48 h expression of L1-G418R in K562 cells and 24 h MMC treatment, n = 3 independent experiments, P values were calculated by a two-tailed unpaired t-test. d, Representative images of γ-H2A.x foci in K562 L1-G418R cells before and after 48 h DOX treatment. Scale bars, 10 μm. e, Representative images of 53BP1 foci in K562 L1-G418R cells before and after 48 h DOX treatment. Scale bar, 10 μm. f, Percentage of K562 L1-G418R cells with more than five foci. Each dot corresponds to an independent experiment. g, Percentage of K562 L1-G418R cells with more than five foci. Each dot corresponds to an independent experiment. h, Image of K562 L1-G418R wild-type and mutant colonies grown in semisold methylcellulose media following DOX treatment and G418 selection. i, L1 retrotransposition measurement using K562 L1-G418R wild-type and mutant cells (sg1 and sg2, two different sgRNAs), n = 18 independent experiments. j, L1 retrotransposition measurement using L1-G418R in K562 cells (wild-type, FANCD2 mutant and FANCD2 complemented cell lines). Each dot represents data from one independent experiment. k, Schema of the pJJ101/L1.3 reporter used in human fibroblasts. l–n, L1 retrotransposition measurement using pJJ101/L1.3 reporter in: PD220 (FANCA−/−) and complemented line (l), PD331 (FANCC−/−) and complemented line (m) and PD20 (FANCD2−/−) and complemented line (n), n = 3 independent experiments, P values were calculated by a two-tailed unpaired t-test. Data represent mean and s.e.m. Unless otherwise specified, P values were calculated by a two-tailed Mann–Whitney U-test.Source data

Fig 5: FA DNA ICL repair factors restrain SN1 LINE-1 retrotransposition during embryogenesis.Quantification of the frequency of SN1 integrations was quantified by ddPCR in E18.5 embryos. Each point represents one embryo. Various tissues with different replicative potentials were assessed in; a) Ercc1−/−, b) Fanca−/−, c) Fancd2−/−, or d) Fancd2-/R E18.5 embryos. e) The expression of SN1 LINE-1 reporter (Sn1 Line-1 Orf2p) was quantified by RT-qPCR in multiple tissues of wildtype and Ercc1−/− adult mice and embryos carrying the SN1 LINE-1 reporter. Expression was normalized to Gapdh and made relative to wildtype E18.5 embryos. n = 3 adult mice per genotype and n = 3 embryos per genotype f) The expression of SN1 LINE-1 reporter (Sn1 Line-1 Orf2p) was quantified by RT-qPCR in multiple tissues of wildtype adult mice and embryos carrying the SN1 LINE-1 reporter. Expression was normalized to Gapdh and made relative to the adult heart. Each point represents one animal. Data represent mean and s.e.m. P values were calculated by two-tailed Mann-Whitney U-test. Source data