FANCM regulates repair pathway choice at stalled replication forks

Abstract

Repair pathway "choice" at stalled mammalian replication forks is an important determinant of genome stability; however, the underlying mechanisms are poorly understood. FANCM encodes a multi-domain scaffolding and motor protein that interacts with several distinct repair protein complexes at stalled forks. Here, we use defined mutations engineered within endogenous Fancm in mouse embryonic stem cells to study how Fancm regulates stalled fork repair. We find that distinct FANCM repair functions are enacted by molecularly separable scaffolding domains. These findings define FANCM as a key mediator of repair pathway choice at stalled replication forks and reveal its molecular mechanism. Notably, mutations that inactivate FANCM ATPase function disable all its repair functions and "trap" FANCM at stalled forks. We find that Brca1 hypomorphic mutants are synthetic lethal with Fancm null or Fancm ATPase-defective mutants. The ATPase function of FANCM may therefore represent a promising "druggable" target for therapy of BRCA1-linked cancer.

Keywords: BRCA1; Bloom’s syndrome helicase; FANCM; Fanconi anemia; break-induced replication; genomic instability; homologous recombination; replication restart; synthetic lethality; tandem duplication.

Figure 1.. FANCM is recruited to Tus/Ter-stalled mammalian replication forks.

See also Figure S1. A. Cartoon of FANCM protein and gene structure. DEAH: helicase domain. B. 6xTer-HR reporter and repair products of Tus/Ter-induced fork stalling. Grey boxes: mutant GFP alleles. Orange triangle: 6xTer array. Blue line: I-SceI restriction site. Ovals A and B: artificial 5’ and 3’ RFP exons. Red ovals: wild type RFP-coding exons. STGC/LTGC: short/long tract gene conversion. TD: tandem duplication. Red zig-zag: non-homologous TD breakpoint. C. Fancm^Δ85 allele, showing frame-shift product with premature stop codon (*). D. Left panel: Immunoblot of chromatin-extracted FANCM in Fancm^+/+ and Fancm^Δ85/Δ clones. H3: Histone H3 loading control. Right panel: loss of FANCM band in siFANCM-treated samples. siLuc: control siRNA to Luciferase. *: background band. E. Immunoblot showing FANCD2 ubiquitination in Fancm^+/+ and Fancm^Δ85/Δ clones in presence or absence of MMC. β-tubulin: loading control. F. Proliferative competition assay in MMC, measuring enrichment of GFP⁺ Fancm^+/+ vs. GFP⁻ Fancm^Δ85/Δ cells. Data shows mean ± standard deviation (SD), n=3. Here and all subsequent growth assays, data normalized to 0 μg/mL MMC. G. ChIP analysis of FANCM at Tus/Ter. Cartoon shows qPCR primer positions for ChIP (red half-arrows; GFP sequence not shown). Numbers indicate distance in bp from outer primer to nearest edge of 6xTer array. Orange triangles: Ter sites. Blue line: I-SceI restriction site. Lower panel: FANCM ChIP 24 hours after transfection with empty vector (EV; gold) or Tus-F140A (purple). Data here and in all subsequent ChIP figures shows mean of 2^−ΔΔCT values, normalized to EV and β-Actin control locus (see STAR methods). Data shows mean ± SD. Analysis by one-way ANOVA (n=3). In this and all subsequent figures: *: P < 0.05: **: P < 0.01; ***: P < 0.001; ****: P < 0.0001; ns: not significant. H. ChIP analysis of FANCM spreading at Tus/Ter or at I-SceI-induced DSB. Data shows mean ± SD (n=3).

Figure 2.. FANCM regulates three distinct pathways of stalled fork repair.

See also Figure S2. A. Tus/Ter-induced repair in Fancm^+/+ (white) clones vs. Fancm^Δ85/Δ (gray) clones. Data shows mean ± SEM. Analysis by one-way ANOVA (n=4). B. I-SceI-induced HR measured in same experiment. C. Representative raw FACS data (uncorrected for transfection efficiency) for Fancm^+/+ and Fancm^Δ85/Δ cells co-transfected with empty vector (EV), I-SceI or Tus and siRNAs as shown. FACS plots pooled from n=4. Numbers show percentages. D. Tus/Ter-induced repair in Fancm^+/+ clone #48 and Fancm^Δ85/Δ85 clone #39 co-transfected with Tus and siRNAs as shown (see STAR Methods). Data shows mean ± SEM. Analysis by Student’s t-test (n=4). E. I-SceI-induced repair measured in parallel in same experiment.

Figure 3.. The FANCM-FA core complex interaction specifically mediates Tus/Ter-induced STGC.

See also Figure S3. A. Fancm^ΔMM1 allele and RT qPCR analysis of MM1 and MM2 encoding mRNA in Fancm^+/− and Fancm^ΔMM1/− cells. Red half-arrows: RT qPCR primers. Here and in all subsequent expression analyses, data normalized to Gapdh mRNA using the 2^−ΔCT method (see STAR methods). Data shows mean ± SD. Analysis by Student’s t-test (n=3). B. Immunoblot of chromatin-extracted FANCM in Fancm^+/− and Fancm^ΔMM1/− clones. *: non-specific band. C. Immunoblot showing FANCD2 ubiquitination in Fancm^+/− and Fancm^ΔMM1/− clones. D. Proliferative competition assay in MMC, measuring enrichment of GFP⁺ Fancm^+/− vs. GFP⁻ Fancm^ΔMM1/− cells. Data shows mean ± SD (n=3). E and F. ChIP analysis of FANCM (E) and FANCA, FANCL and BLM (F) at Tus/Ter in Fancm^+/− and Fancm^ΔMM1/− cells. Data shows mean ± SD. Analysis by one-way ANOVA (n=3). G. Tus/Ter-induced repair in Fancm^+/− (white) clones vs. Fancm^ΔMM1/− (gray) clones. Data shows mean ± SEM. Analysis by one-way ANOVA (n=5). H. Tus/Ter-induced repair in Fancm^+/− vs. Fancm^ΔMM1/− clones co-transfected with Tus and siRNAs as shown. Data shows mean ± SEM. Analysis by Student’s t-test (n=6).

Figure 4.. The FANCM-BLM interaction suppresses LTGC and TD formation at stalled forks.

See also Figure S4. A. Fancm^ΔMM2 allele and RT qPCR analysis of MM1 and MM2 encoding mRNA in Fancm^+/− and Fancm^ΔMM2/−clones. Data shows mean ± SD. Analysis by Student’s t-test (n=3). B. Immunoblot of chromatin-extracted FANCM in Fancm^+/− and Fancm^ΔMM2/− clones. C. Immunoblot showing FANCD2 ubiquitination in Fancm^+/− and Fancm^ΔMM2/− cells. D and E. ChIP analysis of BLM (D), FANCM, FANCA, and FANCL (E) at Tus/Ter in Fancm^+/− and Fancm^ΔMM2/− cells. Analysis by one-way ANOVA (n=3). F. Tus/Ter-induced HR in Fancm^+/− (white) clones vs. Fancm^ΔMM2/− (gray) clones. Data shows mean ± SEM. Analysis by one-way ANOVA (n=4). G. Tus/Ter-induced repair in Fancm^+/− vs. Fancm^ΔMM2/− clones co-transfected with Tus and siRNAs as shown. Data shows mean ± SEM. Analysis by Student’s t-test (n=5).

Figure 5.. FANCM ATP hydrolysis mutants are defective for FANCM-mediated stalled fork repair.

See also Figures S5 and S6. A. Fancm^ΔDEAH allele and RT qPCR analysis of MM2 and DEAH encoding mRNA in Fancm^+/− and Fancm^ΔDEAH/− clones. Data shows mean ± SD. Analysis by Student’s t-test (n=3). B. Immunoblot of chromatin-extracted FANCM in Fancm^+/− and Fancm^ΔDEAH/− clones. C. Immunoblot showing FANCD2 ubiquitination in Fancm^+/− and Fancm^ΔDEAH clones. D-G. ChIP analysis of BLM (D), FANCM (E), FANCA (F) and FANCL (G) at Tus/Ter in Fancm^+/− and Fancm^ΔDEAH/− cells. Data shows mean ± SD. Analysis by one-way ANOVA (n=3). H. Proliferative competition assay in presence of MMC, measuring enrichment of GFP⁺ Fancm^+/− vs. GFP⁻ Fancm^ΔDEAH/−cells (n=3). Error bars: SD. I. Tus/Ter-induced HR in Fancm^+/− (white) clones vs. Fancm^ΔDEAH/− (gray) clones. Data shows mean ± SEM. Analysis by one-way ANOVA (n=5). J. Tus/Ter-induced repair in Fancm^+/− vs. Fancm^ΔDEAH/− clones co-transfected with Tus and siRNAs as shown. Data shows mean ± SEM. Analysis by Student’s t-test (n=5).

Figure 6.. Synthetic lethal interaction between Brca1 and Fancm mutations.

See also Figure S7. A. Left panel: wild type BRCA1 product of Brca1^fl allele. Exon 11-encoded region shown. Cre converts Brca1^fl to Brca1^Δ, with in-frame deletion of exon 11. Right panel: Brca1 exons 10-12 in Brca1^fl allele with PCR primers indicated (red half-arrows). Black triangles: loxP elements. B. Brca1^fl/11 and Brca1^Δ/11 PCR products using primers from panel A. C. RT qPCR analysis of Brca1 Exon 11-encoded mRNA in Brca1^fl/11 and Brca1^Δ/11 clones. Brca1 gene expression level was normalized to Gapdh using the 2^−ΔCT method. Data shows mean ± SEM (n=3). D. Brca1^fl/11 and Brca1^Δ/11 allele recovery in unselected clones following Cre transduction of Brca1^fl\11 cells carrying the Fancm genotypes shown.

Figure 7.. Mechanisms of FANCM in stalled fork repair.

A. FANCM mediates error-free HR (i.e., STGC) at forks bidirectionally arrested at Tus/Ter (orange triangles). FANCM recruits FA core complex, promoting FANCD2 ubiquitination (D2-Ub) and SLX4-mediated incisions (red triangles). FANCM motor function (green dashed arrows) promotes fork remodeling and timely release of FANCM from stalled fork. B. Upper panel: FANCM ΔMM1 mutant retains motor functions but is defective for FA core complex recruitment and FANCD2 ubiquitination, resulting in reduced STGC and increased MMC sensitivity. Lower panel: FANCM ΔDEAH mutant retains FA core complex recruitment and FANCD2 ubiquitination. Defective fork remodeling, possibly combined with FANCM trapping, results in reduced STGC and increased MMC sensitivity. C. FANCM and BLM act in a concerted fashion to suppress aberrant replication fork restart. Hypothetical mechanism of D-loop formation at stalled fork in the absence of a strand exchange step. BLM can dissolve post-replicative double Holliday junction (dHJ). Alternative processing by HJ resolution (red triangles) generates D-loop at stalled fork, with accompanying sister chromatid exchange. Dashed light blue lines: resected nascent lagging strands at stalled fork. FANCM/BLM-mediated branch migration (green and purple dashed arrow) dissociates D-loop, preventing aberrant fork restart. D. Defects in FANCM/BLM interaction allow D-loop to persist, favoring resumption of nascent leading strand synthesis (red half-arrow) with displacement of Tus from Ter (empty orange triangles). Engagement of unknown helicase(s) (red dashed arrow) extends aberrant replication restart by bubble migration. This mechanism is BIR-like but, as shown in C, might not be break-induced. Leftward normal fork duplicates genomic segment ‘a’ bounded, at one end, by the site of fork stalling and, at the other end, by the site of displacement of the restarted leading strand (red). BRCA1 loss impairs DNA end resection, preventing collapse of TD back to single copy by single strand annealing. By default, tandem duplication forms by end joining.