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Fanconi anemia proteins participate in a break-induced-replication-like pathway to counter replication stress

Abstract

Fanconi anemia (FA) is a devastating hereditary disease characterized by bone marrow failure (BMF) and acute myeloid leukemia (AML). As FA-deficient cells are hypersensitive to DNA interstrand crosslinks (ICLs), ICLs are widely assumed to be the lesions responsible for FA symptoms. Here, we show that FA-mutated cells are hypersensitive to persistent replication stress and that FA proteins play a role in the break-induced-replication (BIR)-like pathway for fork restart. Both the BIR-like pathway and ICL repair share almost identical molecular mechanisms of 53BP1–BRCA1-controlled signaling response, SLX4- and FAN1-mediated fork cleavage and POLD3-dependent DNA synthesis, suggesting that the FA pathway is intrinsically one of the BIR-like pathways. Replication stress not only triggers BMF in FA-deficient mice, but also specifically induces monosomy 7, which is associated with progression to AML in patients with FA, in FA-deficient cells.

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Fig. 1: FA-deficient cells are hypersensitive to persistent replication stress.
Fig. 2: FA proteins play a role in the cleavage-coupled BIR pathway.
Fig. 3: BRCA1 and FA proteins coordinate recruitment of SLX4 and FAN1 to chromatin for cleavage of stalled forks.
Fig. 4: SLX4 and FAN1 are required for the cleavage-coupled BIR pathway.
Fig. 5: BRCA1 and 53BP1 antagonistically control FA pathway initiation.
Fig. 6: Persistent replication stress triggers BMF in FancLkf/kf mice.
Fig. 7: Replication stress specifically induces monosomy 7 in FA-deficient cells.
Fig. 8: Models for stalled replication fork restart.

Data availability

All data supporting the findings of this study are available from the corresponding author upon reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank W. Wang for his advice about the paper and antibodies, J. Huang for BRCA1- and FAN1-expressing plasmids and L. Zuo for the SLX4-expressing plasmid. We thank the Imaging Core at the National Center for Protein Sciences at Peking University. This work was supported in part by the Beijing Outstanding Young Scientist Program (BJJWZYJH01201910001005) to Q.L.; the National Natural Science Foundation of China (81672773 and 31870807) to D.X. and Rong Guo; a China Postdoctoral Science Foundation grant (no. 2018M641078) and the National Natural Science Foundation of China (grant no. 31900928) to Y.X.; JSPS KAKENHI (grant no. JP16H06306 to S.T. and JP19H04267 to H.S.) and the JSPS Core-to-Core Program to S.T.

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Contributions

X.X. performed the comet assays, a portion of the drug-sensitivity assays and chromosome spread, the immunoprecipitations, FISH and the mouse experiments. Y.X. performed the immunofluorescence experiments, UFBs staining, most of the drug-sensitivity assays and DNA combing assays and a portion of the MiDAS assays, chromosome spread and the fractionation assays. Ruiyuan Guo and R.X. performed the fractionation assays, a portion of the MiDAS experiments and the DNA combing assays. C.F. generated the plasmids for expressing truncated SLX4 and BRCA1. M.X. performed a portion of the drug-sensitivity assays. H.S. and S.T. generated FANCC-knockout TK6 cells and provided some DT40 knockout cells. M.T. generated most FA-deficient DT40 cells. Q.L., Rong Guo and D.X. designed experiments and interpreted the results. X.X. and D.X. wrote the paper.

Corresponding authors

Correspondence to Yixi Xu or Rong Guo or Dongyi Xu.

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Peer review information Nature Structural & Molecular Biology thanks the anonymous reviewers for their contribution to the peer review of this work. Beth Moorefield was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 FA-deficient cells are hypersensitive to persistent replication stress.

a, Cisplatin sensitivity of the fancC- DT40 cells or chicken FancC (chFancC)-complemented fancC- cells assessed using MTT staining. The mean and s.d. from three independent experiments are shown. b, HU, APH and cisplatin sensitivity of the fancL-/- DT40 cells or human FancL (huFancL)-complemented fancL-/- cells assessed using MTT staining or by colony formation assay. The mean and s.d. from three independent experiments are shown. c, HU sensitivity of the FancL-/- or FancLkf/kf mouse GM by colony formation assay. The mean and s.d. from three independent experiments are shown except the experiment of FancLkf/kf mouse GM in the right panel where n = 2. Statistical source data are provided in Source data.

Source data

Extended Data Fig. 2 Generation of FANCC-/- TK6 cells and FANCA-/- HCT116 cells.

a, Schematics showing FANCC knockout in TK6 cells using CRISPR. b, Western-blotting showing protein level of FANCC in wild-type and FANCC-/- TK6 cells. c, Schematic representation of the generation of FANCA-/- HCT116 cells using CRISPR. Guide sequences are highlighted in blue. PAM sequences are indicated by red lines. The red arrow indicates a putative cleavage site. Red dashes indicate deleted bases. d, Immunoblots showing the expression levels of FANCA in knockout cells. Uncropped images of the are provided in Source data.

Source data

Extended Data Fig. 3 Low doses of HU induce chromosome loss over time, but not a significant increase of chromosome aberration in FANCC-deficient cells.

a, b, Chromosome aberrations in wild-type and FANCC-deficient DT40 (a) and TK6 (b) cells over time. Cells were persistently treated with 100 μM (a) or 40 μM (b) HU. The mean and s.d. from three independent experiments are shown. For each sample from each experiment, 100 metaphase cells were scored. c, Replicated experiments of chromosome number analysis in wild-type and FANCC-/- TK6 cells over time. Cells were cultured with medium containing 40 μM HU for 10 days. 100 metaphases were counted for each sample. Statistical source data are provided in Source data.

Source data

Extended Data Fig. 4 FANCD2 and FANCL act in the same BIR pathway as BRCA1 for restart of stalled forks.

a, Experimental workflows for MiDAS measurement. b, f, Images (left panel) and quantifications (right panel) showing MiDAS (EdU foci, green) rates in FANCD2- (b) and FANCL- (f) depleted BRCA1-/- cells. Scale bar, 5 μm. The mean and s.d. of three independent experiments are shown. c, g, j, Stalled replication fork restart rates in FANCD2- (c), FANCL- (g) and POLD3- (j) depleted wild-type or BRCA1-/- cells. The mean and s.d. from three independent experiments are shown. d, h, Comet assays measuring DSBs in FANCD2- (d) and FANCL- (h) depleted BRCA1-/- cells. The mean and s.e.m. are shown; the numbers of cells examined are indicated and data are representative of three independent experiments. *** P < 0.001, ** P < 0.01, * P < 0.05, ns P > 0.05, two-tailed Student’s t-test. e, i, k, Immunoblots showing the knockdown efficiency of FANCD2 (e), FANCL (i) and POLD3 (k) in wild-type or BRCA1-/- HCT116 cells. Uncropped images of the immunoblots and statistical source data including the precise P values are provided in Source data.

Source data

Extended Data Fig. 5 BRCA1 and SLX4 interact each other through their N-terminal regions.

a, schematic representation of the different BRCA1 deletion mutants (left) and their ability to coimmunoprecipitate with GFP-SLX4 from HEK293 extracts (right). b, c, Immunoprecipitation and Western blotting to assess whether the various deletion mutants of BRCA1 described in (a) coimmunoprecipitated with GFP-SLX4. S protein-FLAG-Streptavidin binding peptide (SFB)-tagged BRCA1 mutants and GFP-SLX4 were co-expressed in HEK293 cells. d-g, Immunoprecipitation and Western blotting to assess whether the various deletion mutants of SLX4 described in Fig. 4b coimmunoprecipitated with BRCA1. SLX4 mutants were fused with a SFB tag and expressed in HEK293. Uncropped images of the immunoblots are provided in Source data.

Source data

Extended Data Fig. 6 ICLs, but not mono-adducts, induce accumulation of DSBs during repair.

Cells were exposed to UV (365-nm, 12 W; 10 cm from the cells) for the indicated times after incubation with psoralen (10 μg/ml) or angelicin (10 μg/ml) for 30 min and were harvested for use in comet assays after 8 h. The mean and s.e.m. are shown; the numbers of cells examined are indicated and data are representative of three independent experiments. **** P < 0.0001, two tailed Student’s t-test. Statistical source data including the precise P values are provided in Source data.

Source data

Extended Data Fig. 7 FANCD2 and POLD3 act in the same pathway to repair ICLs.

a, Cisplatin sensitivity of various DT40 knockout cells. b, Graphic showing the cisplatin and MMC sensitivity of POLD3-depleted PD20 or FANCD2-complemented PD20 cells. The mean and s.d. from three independent experiments are shown. Statistical source data are provided in Source data.

Source data

Extended Data Fig. 8 Generation of FancL knockout mice.

a, Schematic representation of the wild-type and targeted genomic DNA in the FancL gene. Arrows indicate locations targeted by primers for genomic PCR. b, c, Genomic PCR analysis to show that exon 6 is replaced by a knockout-first cassette or is undetectable in the FancLkf/kf (b) or FancL-/- (c) mice. d, mRNA level of FancL in FancLkf/kf mice. The mean and s.d. from three independent experiments are shown. e, Observed and expected birth numbers of FancLkf/kf mice. f, Weight of wild-type and FancLkf/kf mice at 3 or 8 weeks of age. The mean and s.d. are shown. * P < 0.05, ns P > 0.05, two tailed Student’s t-test; n = 10 biologically independent mice. Uncropped images of the gels and statistical source data including the precise P values are provided in Source data.

Source data

Extended Data Fig. 9 Chromosome number analysis after MMC or APH treatment.

Cells were persistently treated with APH (40 nM for 6 days) or pulsed with MMC (40 ng/mL for 1 day). 100 metaphases for each sample in each independent experiment were counted. Experiments were replicated three times. These experiments were carried out together with that in Fig. 1f and the same control samples were used. Statistical source data are provided in Source data.

Source data

Supplementary information

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Xu, X., Xu, Y., Guo, R. et al. Fanconi anemia proteins participate in a break-induced-replication-like pathway to counter replication stress. Nat Struct Mol Biol 28, 487–500 (2021). https://doi.org/10.1038/s41594-021-00602-9

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