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. 2014 Aug 1;33(15):1698-712.
doi: 10.15252/embj.201387530. Epub 2014 Jun 25.

Crosstalk between BRCA-Fanconi anemia and mismatch repair pathways prevents MSH2-dependent aberrant DNA damage responses

Min Peng  1 , Jenny Xie  1 , Anna Ucher  2 , Janet Stavnezer  2 , Sharon B Cantor  3
Affiliations

Crosstalk between BRCA-Fanconi anemia and mismatch repair pathways prevents MSH2-dependent aberrant DNA damage responses

Min Peng et al. EMBO J. .

Abstract

Several proteins in the BRCA-Fanconi anemia (FA) pathway, such as FANCJ, BRCA1, and FANCD2, interact with mismatch repair (MMR) pathway factors, but the significance of this link remains unknown. Unlike the BRCA-FA pathway, the MMR pathway is not essential for cells to survive toxic DNA interstrand crosslinks (ICLs), although MMR proteins bind ICLs and other DNA structures that form at stalled replication forks. We hypothesized that MMR proteins corrupt ICL repair in cells that lack crosstalk between BRCA-FA and MMR pathways. Here, we show that ICL sensitivity of cells lacking the interaction between FANCJ and the MMR protein MLH1 is suppressed by depletion of the upstream mismatch recognition factor MSH2. MSH2 depletion suppresses an aberrant DNA damage response, restores cell cycle progression, and promotes ICL resistance through a Rad18-dependent mechanism. MSH2 depletion also suppresses ICL sensitivity in cells deficient for BRCA1 or FANCD2, but not FANCA. Rescue by Msh2 loss was confirmed in Fancd2-null primary mouse cells. Thus, we propose that regulation of MSH2-dependent DNA damage response underlies the importance of interactions between BRCA-FA and MMR pathways.

Keywords: FANCJ; Fanconi anemia; MLH1; mismatch repair; replication stress.

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Figures

Figure 1
Figure 1. Sensitivity to mitomycin C (MMC) in FANCJ-deficient cells is suppressed by loss of MSH2, but not MLH1, DNA-PKcs, or 53BP1

  1. Immunoblot analysis of FANCJ, MLH1, and MSH2 expressions in U2OS cells treated with indicated siRNAs. β-actin was used as a loading control.

  2. Graph shows the percentage of viable cells 5 days after indicated dose of MMC.

  3. Immunoblot analysis of FANCJ and MSH2 expressions in human MSH2-null (HEC59) and MSH2-proficeint (HEC59+Chr2) cell lines treated with indicated siRNA reagents to Con or FANCJ (a or b).

  4. Graph shows the percentage of viable cells 5 days after 500 nM MMC.

  5. Immunoblot analysis of FANCJ and DNA-PKcs in DNA-PKcs-deficient (M059J) and DNA-PKcs-proficient (M059K) cells treated with siRNA reagents to Con or FANCJ (a or b).

  6. Graph shows the percentage of viable cells 5 days after 250 nM MMC.

  7. Immunoblot analysis showing 53BP1 or FANCJ expression in U2OS cells stably expressing shRNA vectors to either control or 53BP1 (a or b) that were also transfected with siRNAs to Con or FANCJ.

  8. Graph shows the percentage of viable cells 5 days after 250 nM MMC.

Data information: Where shown, error bars represent standard deviations from three independent experiments.
Figure 2
Figure 2. Mitomycin C (MMC)-induced sensitivity in cells lacking the FANCJ–MLH1 interaction is suppressed by MSH2 depletion
A Immunoblot analysis of FANCJ and/or MLH1 in the indicated Fanconi anemia (FA)-J complemented cell lines from whole-cell extracts or following immunoprecipitation. B Graph shows the percentage of viable cells 5 days after increasing doses of MMC. C Immunoblot analysis of FANCJ, MSH2, and MLH1 expressions in the FA-J cell lines treated with the indicated siRNAs. D, E Graphs show the percentage of viable cells 5 days after increasing doses of MMC (D) or as compared at one dose (E). Data information: Where shown, error bars represent standard deviations from three independent experiments. The asterisk (*) represents a P-value < 0.01.
Figure 3
Figure 3. Aberrant DNA damage responses in cells lacking the FANCJ–MLH1 interaction are suppressed by MSH2 depletion

  1. Immunoblot analysis of FANCJ and MSH2 expressions in the Fanconi anemia (FA)-J cell lines expressing indicated shRNAs.

  2. Representative cell cycle profiles based on PI staining of DNA content for the indicated FA-J cell lines untreated (U) or at the indicated times following 0.25 μg/ml melphalan treatment.

  3. MSH2 depletion reduces DNA-PKcs Ser2056 phosphorylation after mitomycin C (MMC) treatment. Green fluorescent protein (GFP) expression indicates shRNA vector-infected FANCJK141/142A FA-J cells. Representative immunofluorescence images are shown.

  4. Immunoblot analysis with indicated antibodies.

  5. Genomic instability is suppressed by MSH2 depletion after 250 nM MMC for 16 h. Representative metaphases show examples of (a) broken and (b) quad-radial chromosomes that were suppressed by MSH2 depletion.

  6. Graph shows number of breaks and radials quantified from 50 metaphases.

Data information: Where shown, error bars represent standard deviations from three independent experiments. The asterisk (*) represents a P-value < 0.01.
Figure 4
Figure 4. MSH2 depletion does not enhance RAD51 foci, but suppresses mitomycin C (MMC) sensitivity through a Rad18-dependent mechanism
A Immunoblot analysis of MSH2 expression in the FANCJK141/142A Fanconi anemia (FA)-J cells treated with the indicated shRNAs. B Quantification of the percentage of γ-H2AX foci-positive cells that have also RAD51 foci after 250 nM MMC treatment for 16 h. C, D Immunoblot analysis with the indicated Abs of chromatin fractions from FANCJK141/142A FA-J cells stably expressing shRNA to Con or MSH2. E Immunoblot analysis of Rad18 and MSH2 expressions in FANCJK141/142A FA-J cells stably expressing shRNA to Con or MSH2 treated with indicated siRNAs. F Graph shows the percentage of viable cells 5 days after 125 nM MMC. Data information: Where shown, error bars represent standard deviations from three independent experiments. The asterisk (*) represents a P-value < 0.01.
Figure 5
Figure 5. Fanconi anemia (FA)-J cells lacking the FANCJ–MLH1 interaction have a pronounced cell cycle progression defect that is suppressed by MSH2 depletion
A Immunoblot analysis of phosphorylation of DNA-PKcs in the FA-J cell lines before or after aphidicolin (APH) treatment and release. B, C Immunofloresence representative figure (B) and quantification (C) after 18 h APH (3 μg/ml) treatment and 1 h release in the presence of EdU shows the FA-J cell lines differ in their ability to restore DNA synthesis. Over 300 cells per experiment were counted. D Graph shows the percentage of cells with 4N DNA content after indicated time following APH release. E MSH2 deletion suppresses the APH-induced cell cycle progression defect in FANCJK141/142A expressing FA-J cells. Graph shows the percent cells with 4N DNA content 24 h following APH release in shRNA-treated cell lines.
Figure 6
Figure 6. MSH2 depletion suppresses mitomycin C (MMC) sensitivity of cells deficient for FANCD2 or BRCA1, but not FANCA, and rescue correlates with reduced DNA-PKcs phosphorylation in FANCD2-deficient cells
A Immunoblot analysis of FANCJ, FANCD2, BRCA1, FANCA, and MSH2 expressions in U2OS cells treated with indicated siRNA. B Graph shows the percentage of viable cells 5 days after 500 nM MMC. The asterisk (*) represents a P-value < 0.01. C, D Fanconi anemia (FA)-D2 (FANCD2-null) (PD20) patient cells stably expressing shRNA vectors with green fluorescent protein (GFP; control) or MSH2 were treated with 250 nM MMC for 16 h or left untreated. GFP expression indicates shRNA vector-infected cells. Representative immunofluorescence images (C) and quantification (D) of the percentage of cells with DNA-PKcs foci after 250 nM MMC treatment for 16 h. E Immunoblot analysis with the indicated Abs of chromatin fractionated FA-D2 cells stably expressing shRNA to Con or MSH2. Data information: Where shown, error bars represent standard deviations from three independent experiments.
Figure 7
Figure 7. Sensitivity to mitomycin C (MMC) and the aberrant DNA damage response in Fancd2-null mouse cells are suppressed by Msh2 deletion
A Chart shows embryos obtained from indicated cross. B Graph shows the percentage of viable primary mouse embryonic fibroblasts (MEFs) with the designated genotypes 5 days after MMC treatment. Three independent MEFs per genotype were analyzed. C, D Representative immunofluorescence images (C) and quantification (D) of cells with foci positive for the ATM/ATR substrate phosphorylation and γ-H2AX following 250 nM MMC treatment. E, F Representative immunofluorescence images (E) and quantification (F) of cells with 53BP1 foci and γ-H2AX in untreated MEFs. Data information: Where shown, error bars represent standard deviations from three independent experiments. The asterisk (*) represents a P-value < 0.01.
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