The structure-specific endonuclease complex SLX4-XPF regulates Tus-Ter-induced homologous recombination

Abstract

Vertebrate replication forks arrested at interstrand DNA cross-links (ICLs) engage the Fanconi anemia pathway to incise arrested forks, 'unhooking' the ICL and forming a double strand break (DSB) that is repaired by homologous recombination (HR). The FANCP product, SLX4, in complex with the XPF (also known as FANCQ or ERCC4)-ERCC1 endonuclease, mediates ICL unhooking. Whether this mechanism operates at replication fork barriers other than ICLs is unknown. Here, we study the role of mouse SLX4 in HR triggered by a site-specific chromosomal DNA-protein replication fork barrier formed by the Escherichia coli-derived Tus-Ter complex. We show that SLX4-XPF is required for Tus-Ter-induced HR but not for error-free HR induced by a replication-independent DSB. We additionally uncover a role for SLX4-XPF in DSB-induced long-tract gene conversion, an error-prone HR pathway related to break-induced replication. Notably, Slx4 and Xpf mutants that are defective for Tus-Ter-induced HR are hypersensitive to ICLs and also to the DNA-protein cross-linking agents 5-aza-2'-deoxycytidine and zebularine. Collectively, these findings show that SLX4-XPF can process DNA-protein fork barriers for HR and that the Tus-Ter system recapitulates this process.

Extended Data Fig. 1. Characterization of Slx4^Δ125/Δ cells

a. RT-qPCR analysis of Slx4 mRNA normalized to Gapdh mRNA using the 2^–ΔCT method in three independent experiments. Each data point is an average of three technical replicates. Data shows mean of three independent biological replicates (n=3), Error bars: standard deviation (s.d.) Statistical analysis using Student’s t-test. b and c. Ratio of Tus/Ter-induced (b) and I-SceI-induced (c) LTGC: total HR in Slx4^+/+ clones and Slx4^Δ125/Δ clones. Data shows mean values, n=6. Error bars: s.e.m. Analysis by ANOVA. P-value *p < 0.05 and ****p < 0.0001 d. CRISPR/Cas9 with dual sgRNA targeting to generate Slx4^+/− hemizygous cells. The Slx4⁻ allele contains a 19.3kb deletion between exons 2 and 15. Red half arrowheads: PCR and sequencing primers specific to Exons 2 and 15. Predicted PCR product sizes for Slx4⁺ (wild type) allele shown. e. Gel shows PCR products detected using gDNA from Slx4^+/+ and Slx4^+/− clones. Note the 444 bp PCR product formed across the 19.3kb deletion between exons 2 and 15 in Slx4^+/− cells. f. DNA sequencing chromatogram of PCR products from (e). g. RT qPCR analysis of Slx4 mRNA in Slx4^+/+ and Slx4^+/− cells. Data shows mean of three independent experiments (n=3) normalized to Gapdh mRNA using the 2^–ΔΔCT method and analyzed by unpaired Student’s t-test. Error bars: s.d.. h. and i. Quantification of colony formation of Slx4^+/+ vs. Slx4^+/− clones in the presence of MMC (h), Olaparib (i). Data shows mean of three biologically independent replicates, n=3. Error bars: s.d. j. Tus/Ter-induced and I-SceI-induced repair frequencies in Slx4^+/+ and Slx4^+/− cells. Data shows mean values of five biologically independent replicates, n=5. Error bars: s.e.m. Statistical analysis using ANOVA.

Extended Data Fig. 2. The SLX4 UBZ domain promotes resistance to DPC-inducing drugs

a. Strategy for in-frame deletion of UBZ domain-encoding regions of Slx4. Red half-arrow heads: genotyping PCR primers. Gel shows PCR products using gDNA from Slx4^+/− and Slx4^ΔUBZ/− clones. Sequencing chromatogram shows in-frame breakpoint of Slx4^ΔUBZ allele. b. RT-qPCR analysis of mRNA encoding UBZ, MLR, SAP and SBD domains in Slx4^+/− and Slx4^ΔUBZ/−clones. Data normalized to Gapdh mRNA using the 2^–ΔCT method, mRNA expression in Slx4^ΔUBZ/− samples were normalized to Slx4^+/− of the same experiment. Each data point is an average of three technical replicates. Data shows mean of three independent biological replicates (n=3). Analysis by unpaired Student’s t-test (n=3). Error bars: standard deviation. P=0.0125 for mRNA expression of the UBZ region in Slx4^+/− compared to Slx4^ΔUBZ/−. No significant differences were observed in expression levels of all other domains. c-h. Quantification of colony formation of Slx4^+/−, Slx4^ΔUBZ/− and Brca1-Δexon11 clones in the presence of MMC (c), Olaparib (d), 5-aza-2′-deoxycytidine (e) Zebularine (f), Hydroxyurea (g) and Camptothecin (h). Data shows mean of values of three biologically independent replicates, n=3. Analysis using Student’s t-test. P-value *p < 0.05 and ***p < 0.001. Red asterisks refer to comparison between Slx4^+/− and Slx4^ΔUBZ/− clones; blue asterisks denote comparison between Slx4^+/− and Brca1-Δexon11 clones and blue bracket with red asterisks denotes comparison between Slx4^ΔUBZ/− and Brca1-Δexon11 clones. Error bars: s.d. i. RT-qPCR analysis of N-terminal Flag-tagged wild-type full-length human SLX4 and UBZ 4C>A expression plasmids 48h after transfection. Data normalized to Gapdh mRNA using the 2^–ΔCT method and compared to empty vector (EV) control. Each data point is an average of three technical replicates. Data shows mean of three independent biological replicates (n=3). Error bar: s.d. j. Western blot analysis of the chromatin-bound fraction of various human 3xFLAG-SLX4 full-length and UBZ 4C>A transiently expressed for 48 hours and blotted with anti-FLAG antibody. k. Western blot analysis of whole cell extract (WCE) and chromatin-bound fraction of clones of Slx4^+/− and Slx4^ΔUBZ/− tagged with a dual degron containing a C-terminal 8xHA tag and untagged hemizygote cells (+/–). Asterisk (*) denotes non-specific bands.

Extended Data Fig. 3. The SLX4 UBZ domain mediates I-SceI-induced LTGC

a and b. Tus/Ter-induced LTGC (a) and LTGC/Total HR ratio (b) in Slx4^+/− and Slx4^ΔUBZ/− clones. Data shows mean values of five biologically independent replicates, n=5. Error bars: s.e.m. Analysis by one-way ANOVA. P-value: ****p < 0.0001. Error bars: s.e.m. c. Impact of exogenous hSLX4 on Tus/Ter-induced LTGC in two independent Slx4^+/− and two independent Slx4^ΔUBZ/− clones. EV: empty vector. 4C>A: hSLX4 with inactivated UBZ motifs. Data shows mean values of five biologically independent replicates, n=6. Error bars: s.e.m. Analysis by unpaired Student’s t-test. Error bars: s.e.m. d and e. I-SceI-induced LTGC (d) and LTGC/Total HR ratio (e) in Slx4^+/− and Slx4^ΔUBZ/− clones. Data shows mean values of five biologically independent replicates, n=5. P-value: ****p < 0.0001 Error bars: s.e.m. Analysis by one-way ANOVA. Error bars: s.e.m. f. Impact of exogenous hSLX4 on I-SceI-induced LTGC in two independent Slx4^+/− and two independent Slx4^ΔUBZ/− clones. EV: empty vector. 4C>A: hSLX4 with inactivated UBZ motifs. Data shows mean values of six biologically independent replicates, n=6. Error bars: s.e.m. Analysis by unpaired Student’s t-test. P-value: *p < 0.05. Error bars: s.e.m.

Extended Data Fig. 4. Characterization of Slx4^ΔSAP/− and Slx4^ΔSBD/− mutant clones

a. Strategy for in-frame deletion of SAP domain-encoding region of Slx4. Red half-arrow heads: genotyping PCR primers. Gels show PCR products using gDNA from Slx4^+/− and Slx4^ΔSAP/− clones. Sequencing chromatogram shows in-frame breakpoint of Slx4^ΔSAP allele. b. RT-qPCR analysis of UBZ, MLR, SAP and SBD domains encoding mRNA in Slx4^+/− and Slx4^ΔSAP/−clones. Data normalized to Gapdh mRNA using the 2^–ΔCT method, mRNA expression in Slx4^ΔSAP/− were normalized to Slx4^+/−. Each data point is an average of three technical replicates. Data shows mean of three independent biological replicates (n=3). Analysis by unpaired Student’s t-test. Error bars: s.d. P=0.0478 for mRNA expression of the SAP region in Slx4^+/− compared to Slx4^ΔSAP/−. No significant differences were observed in expression levels of all other domains. c. Anti-HA immunoblot of two independent HA-degron tagged Slx4^ΔSAP/− and Slx4^+/− isogenic cell lines. The parental Slx4^+/− untagged cell line was used as a control. d and e. Tus/Ter-induced LTGC (d) and I-SceI-induced LTGC (e) in Slx4^+/− and Slx4^ΔSAP/− clones. Data shows mean values of five biologically independent replicates, n=5. Error bars: s.e.m. Analysis by ordinary one-way ANOVA. Error bars: s.e.m. f. Strategy for in-frame deletion of SBD domain-encoding region of Slx4. Red half-arrow heads: genotyping PCR primers. Gels show PCR products using gDNA from Slx4^+/− and Slx4^ΔSBD/− clones. Sequencing chromatogram shows in-frame breakpoint of Slx4^ΔSBD allele. g. RT-qPCR analysis of UBZ, MLR, SAP and SBD domains encoding mRNA in Slx4^+/− and Slx4^ΔSBD/−clones. Data normalized to Gapdh mRNA using the 2^–ΔCT method, mRNA expression in Slx4^ΔSBD/− was normalized to Slx4^+/− of the same experiment. Each data point is an average of three technical replicates. Data shows mean of three independent biological replicates (n=3). Analysis by unpaired Student’s t-test (n=3). Error bars: standard deviation. P=0.048 for mRNA expression of the SAP region in Slx4^+/− compared to Slx4^ΔSBD/−. No significant differences were observed in expression levels of all other domains. h. Anti-HA immunoblot of two independent HA-degron tagged Slx4^ΔSBD/− and Slx4^+/− isogenic cell lines. The parental Slx4^+/− untagged cell line was used as a control. i and j. Tus/Ter-induced LTGC (i) and I-SceI-induced LTGC (j) in Slx4^+/− and Slx4^ΔSBD/− clones. Data shows mean values of five biologically independent replicates, n=5. Error bars: s.e.m. Analysis by ordinary one-way ANOVA. Error bars: s.e.m.

Extended Data Fig. 5. Slx4^ΔSAP/− and Slx4^ΔSBD/− mutant clones are resistant to DPC-inducing drugs

a-f. Quantification of colony formation of Slx4^+/− vs. Slx4^ΔSAP/− clones in the presence of MMC (a), Olaparib (b), 5-aza-2′-deoxycytidine (c), Zebularine (d) Hydroxyurea (e) and Camptothecin (f). Data show mean of three biologically independent replicates normalized to untreated samples (n=3). Error bars: s.d. g-l. Quantification of colony formation of Slx4^+/− vs. Slx4^ΔSBD/− clones in the presence of MMC (g), Olaparib (h), 5-aza-2′-deoxycytidine (i), Zebularine (j) Hydroxyurea (k) and Camptothecin (l). Data show mean of three biologically independent replicates normalized to untreated samples (n=3). Error bars: s.d.

Extended Data Fig. 6. Characterization of Slx4^ΔMLR/− mutant clones

a. Strategy for in-frame deletion of MLR domain-encoding region of Slx4. Red half-arrow heads: genotyping PCR primers. Gels show PCR products using gDNA from Slx4^+/− and Slx4^ΔMLR/− clones. Sequencing chromatogram shows in-frame breakpoint of Slx4^ΔMLR allele. b. RT-qPCR analysis of UBZ, MLR, SAP and SBD domains encoding mRNA in Slx4^+/− and Slx4^ΔMLR/−clones. Data normalized to Gapdh mRNA using the 2^–ΔCT method, mRNA expression in Slx4^ΔMLR/− were normalized to Slx4^+/− of the same experiment. Each data point is an average of three technical replicates. Data shows mean of three independent biological replicates (n=3). Analysis by unpaired Student’s t-test (n=3). Error bars: standard deviation (s.d.). P=0.01 for mRNA expression of the MLR region in Slx4^+/− compared to Slx4^ΔMLR/−. No significant differences were observed in expression levels of all other domains. c Anti-HA immunoblot of two independent HA-degron tagged Slx4^ΔMLR/− and Slx4^+/− isogenic cell lines. The parental Slx4^+/− untagged cell line was used as a control. d-g. Quantification of cell survival assays of Slx4^+/− and Slx4^ΔMLR/− clones in the presence of MMC (d), Olaparib (e) 5-aza-2′-deoxycytidine (f) Zebularine (g), Hydroxyurea (h) and Camptothecin (i). Data show mean values of three biologically independent replicates, n=3. P-value: *p < 0.05. Analysis using unpaired Student’s t-test. Error bars: s.d. j. Representative images of metaphase spreads showing chromatid breaks (black arrows) and radial chromosomes (black arrowheads) in cells either untreated or treated with 20 ng/mL MMC for 12h in Slx4^ΔSAP/− and Slx4^ΔSBD/− cells. k. and l. Quantitation of the number of breaks (k) and radial chromosomes (l) per metaphase nucleus in Slx4 mutant cells shown. Treated and untreated cells were harvested at the same time and each sample represented one independent experiment, n=1. Data shows mean values of breaks or radials per genotype. Analysis using unpaired Student’s t-test. P-value: ****p < 0.0001.

Extended Data Fig. 7. The SLX4 MLR domain contributes to I-SceI-induced LTGC

a and b. Tus/Ter-induced LTGC (a) and I-SceI-induced LTGC (b) in Slx4^+/− and Slx4^ΔMLR/− clones. Data shows mean values of five biologically independent replicates, n=5. P-value: **p < 0.01. Error bars: s.e.m. Analysis by ordinary one-way ANOVA. Error bars: s.e.m. c and d. Impact of exogenous hSLX4 alleles (see Fig. 5c) on Tus/Ter-induced LTGC (c) and on I-SceI-induced LTGC (d) in Slx4^+/− and Slx4^ΔUBZ/− clones. EV: empty vector. 4C>A: hSLX4 with inactivated UBZ motifs. Data shows mean values of six biologically independent replicates, n=6. P-value: *p < 0.05; **p < 0.01; ***p < 0.00. Error bars: s.e.m. Analysis by unpaired Student’s t-test.

Extended Data Fig. 8. Characterization of Xpf^+/− and Xpf^ΔNuc/− cells

a. CRISPR/Cas9 with dual sgRNA strategy for generation of Xpf^+/− hemizygous cells. The Xpf⁻ allele contains a 42.3 kb deletion between the 5’UTR and exon 11. Red half arrowheads: PCR and sequencing primers specific to 5’UTR and Exon 11. Predicted PCR product sizes for Xpf⁺ (wild type) allele shown. b. Gel shows PCR products using gDNA from Xpf^+/+ and Xpf^+/− clones. Note the 440 bp PCR product formed across the 42.3kb deletion between the 5’UTR and exon 11 in Xpf^+/− cells. c. RT qPCR analysis of mRNA in Xpf^+/+ and Xpf^+/− cells. Data shows mean of three independent experiments (n=3) normalized to Gapdh mRNA using the 2^–ΔΔCT method and analyzed by unpaired Student’s t-test. Error bars: s.d. d. Western blot of whole cell lysates in Xpf^+/+ or Xpf^+/− clones. siLuc and siErcc1 were compared in Xpf^+/− cell line 48 hours after transfection to validate specificity of the Ercc1 band. e. and f. Quantification of colony formation of Xpf^+/+ vs. Xpf^+/− clones in the presence of MMC (e), Olaparib (f). Data show mean values of three biologically independent replicates, n=3. Error bars: s.d. g. Tus/Ter-induced and I-SceI-induced HR frequencies in Xpf^+/+ or Xpf^+/− clones. Data shows mean values of four biologically independent replicates, n=4. Error bars: s.e.m. Analysis by unpaired Student’s t-test. Error bars: s.e.m h. RT-qPCR analysis of mRNA encoding the XPF helicase, nuclease and Hh2h domains in 4 isogenic Xpf ^+/− clones and 4 Xpf ^ΔNuc/− clones. Data normalized to Gapdh mRNA using the 2^–ΔCT method, mRNA expression in Xpf ^ΔNuc/− was normalized to Xpf ^+/− of the same experiment. Each data point is an average of three technical replicates. Data shows mean of three independent biological replicates (n=3). Analysis by unpaired Student’s t-test. Error bars: s.d. P=0.0153 for mRNA expression of the Nuclease region in Xpf ^+/− compared to Xpf ^ΔNuc/−. No significant differences were observed in expression levels of all other domains. i. Western blot analysis of ERCC1 in whole cell extract (WCE). *: non-specific band. β-tubulin: Loading control. j. Western blot analysis of ERCC1 in chromatin fraction of Xpf ^+/− and Xpf ^ΔNuc/− clones. H3: Histone H3 loading control. Note presence of ERCC1 signal in in-frame deleted Xpf ^ΔNuc/− clone #1, and absence of ERCC1 signal in frame-shifted Xpf ^ΔNuc/− clones #2, 11 and 13 k. Cell cycle distribution of Xpf^+/− clone #6 and Xpf^ΔNuc/− clone #1, either untreated or treated with 20, 30, 40 or 50 ng/mL of MMC. Data shows mean values of three biologically independent replicates, n=3. P-value: **p < 0.01; ***p < 0.001; ****p < 0.0001. Error bars: s.e.m. Untreated samples were compared using the unpaired Student’s t-test; MMC-treated groups were compared by one-way ANOVA. l and m. Tus/Ter-induced LTGC (l) and I-SceI induced LTGC (m) in Xpf^+/− and Xpf^ΔNuc/− clones. Data shows mean values of six biologically independent replicates, n=6. Error bars: s.e.m. Analysis by ordinary one-way ANOVA. P-value: ****p < 0.0001.

Fig. 1.. Slx4 regulates error-free HR at Tus/Ter stalled forks.

a. Schematic of 6xTer-HR reporter and repair products of Tus/Ter induced fork stalling. Light blue boxes: mutant GFP alleles. Green box: wild type GFP. Open circles A and B: 5’ and 3’ artificial RFP exons, respectively. 5′ Tr-GFP: 5′-truncated GFP. Red triangle: 6xTer array. Black line: I-SceI restriction site. STGC/LTGC, short/long tract gene conversion outcomes. LTGC generates wild-type RFP through RNA splicing (red filled circles). b. Strategy for generation of Slx4^Δ125 frameshift allele. Gel shows products of PCR of gDNA from Slx4^+/+ and Slx4^Δ125/Δ clones. Red letters above sequencing chromatogram indicate frame shift. c. Representative images of colony formation assays performed in the presence of varying concentrations of Mitomycin C (MMC), in Slx4^+/+ (#3) (top) and one Slx4^Δ125/Δ (#40) (middle) cell lines. Right panel: Quantitation of above-mentioned colony formation assays, showing mean of three biologically independent replicates (n=3). Analysis using unpaired Student’s t-test (n=3). P-value: **p < 0.01 and ****p < 0.0001. Error bars: s.d. d. Representative raw FACS data (uncorrected for transfection efficiency) for one Slx4^+/+ (#3) and one Slx4^Δ125/Δ (#40) 6x Ter-HR reporter clone co-transfected with either empty vector (EV), I-SceI or Tus expression vectors as shown. FACS plots produced from pooled technical duplicates, n=6. Numbers indicate percentages. e and f. Tus/Ter-induced STGC (e) and LTGC (f) in Slx4^+/+ clones and Slx4^Δ125/Δ clones. g and h. I-SceI-induced STGC (g) and LTGC (h) in Slx4^+/+ vs. Slx4^Δ125/Δ clones. Data in (e-h) shows mean values of n=6 biologically independent replicates. Error bars: standard error of the mean (s.e.m). Statistical analysis by ANOVA. In this and all subsequent figures: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant.

Fig. 2.. The SLX4 UBZ domain mediates Tus/Ter-induced STGC by retaining SLX4 at stalled forks.

a. Schematic of SLX4 protein. MLR: XPF-interacting domain. SAP: MUS81-interacting domain. SBD: SLX1-interacting domain. Lower panel: SLX4 ΔUBZ protein. b. Cell cycle distribution of Slx4^+/− and Slx4^ΔUBZ/− cells either untreated or treated with 20, 30, 40 or 50 ng/mL of MMC. Data shows mean values of three biologically independent replicates, n=3. Error bars: s.e.m. Untreated samples were compared using unpaired Student’s t-test; MMC-treated groups were compared by one-way ANOVA. P-value: **p < 0.01; ***p < 0.001. c and d. Tus/Ter-induced STGC (c) and I-SceI induced STGC (d) in Slx4^+/− vs. Slx4^ΔUBZ/− clones. Data shows mean values of five biologically independent replicates. Statistical analysis by ANOVA, n=5. P-value: ****p < 0.0001. Error bars: s.e.m. e and f. Impact of exogenous hSLX4 on Tus/Ter induced STGC (e) and I-SceI induced STGC (f) in two independent Slx4^+/− and two independent Slx4^ΔUBZ/− clones. EV: empty vector. 4C>A: hSLX4 with inactivated UBZ motifs. Data shows mean values of six biologically independent replicates. Statistical analysis by unpaired Student’s t-test, n=6. P-value: : **p < 0.01. Error bars: s.e.m. g. Schematic of Slx4^+/−and Slx4^ΔUBZ/− tagged with a dual degron containing a C-terminal 8xHA tag. h. RT-qPCR analysis of Slx4 mRNA in Slx4^deg/− and Slx4^ΔUBZdeg/− clones. Data normalized to Gapdh mRNA using the 2^–ΔCT method. Expression of Slx4^ΔUBZdeg/− normalized to Slx4^deg/−. Each data point is an average of three technical replicates. Data shows mean of three independent biological replicates. Analysis by unpaired Student’s t-test (n=3). Error bars: standard deviation (s.d.). i. Anti-HA immunoblot of HA-degron tagged SLX4, 24 hours after addition of degron-activating drugs 5-IAA and/or Asv. Asv: Asunaprevir. 5-IAA: 5-adamantyl-IAA. H3: Histone H3 loading control. j. Anti-HA ChIP analysis of SLX4-HA at Tus/Ter RFB in Slx4^deg/− and Slx4^ΔUBZdeg/− cells. Each data point is an average of three technical replicates. Data shows mean of three independent biological replicates (n=3). Analysis by unpaired Student’s t-test. P-value: ****p < 0.0001. Error bars: s.d.

Fig. 3.. Interactions of SLX4 with MUS81 and with SLX1 are dispensable for Tus/Ter-induced HR.

a. Schematic of SLX4 protein lacking the MUS81-interacting SAP domain. b and c. Tus/Ter-induced STGC (b) and I-SceI induced STGC (c) in Slx4^+/− vs. Slx4^ΔSAP/− clones. Data shows mean values of five biologically independent replicates. Statistical analysis using ordinary one-way ANOVA, n=5. Error bars: s.e.m. d. Schematic of SLX4 protein lacking the SLX1-interacting SBD domain. e and f. Tus/Ter-induced STGC (e) and I-SceI induced STGC (f) in Slx4^+/− vs. Slx4^ΔSBD/− clones. Data shows mean values of five biologically independent replicates. Statistical analysis by ordinary one-way ANOVA, n=5. Error bars: s.e.m.

Fig. 4.. Loss of the SLX4-XPF interaction causes a characteristic FA phenotype

a. Schematic of SLX4 protein lacking the XPF-interacting MLR domain. b. Cell cycle distribution of Slx4^+/− and Slx4^ΔMLR/− cells either untreated or treated with 20, 30, 40 or 50 ng/mL of MMC. Data shows mean of three biologically independent replicates, n=3. Error bars: s.e.m. Untreated samples were compared using the unpaired Student’s t-test; MMC-treated groups were compared by one-way ANOVA. P-value: *p < 0.05; ***p < 0.001; ****p < 0.0001. c. Representative images of metaphase spreads from Slx4 mutants indicated, showing chromatid breaks (black arrows) and radial chromosomes (black arrowheads) in untreated cells and cells treated with 20 ng/mL MMC for 12h (d) and (e). Treated and untreated cells were harvested at the same time and each sample represented one independent experiment, n=1. Error bars: s.d. Quantitation of the number of breaks (d) and radial chromosomes (e) per metaphase nucleus in Slx4^+/−, Slx4^ΔUBZ/− and Slx4^ΔMLR/− cell lines. Analysis using unpaired Student’s t-test. P-value: : *p < 0.05; **p < 0.01; ****p < 0.0001.

Fig. 5.. The SLX4-XPF interaction is required for Tus/Ter-induced STGC.

a and b Tus/Ter-induced STGC (a) and I-SceI induced STGC (b) in Slx4^+/− vs. Slx4^ΔMLR/− clones. Data shows mean values of five biologically independent replicates, n=4. Analysis by one-way ANOVA, P-value; ****p < 0.0001. Error bars: s.e.m. c. Schematic showing mutants of human SLX4 used for transient expression experiments. d. RT-qPCR analysis of expression of human SLX4 variants in mES cells. SLX4 expression normalized to Gapdh and displayed as fold difference compared to empty vector (EV) of the same experiment. Each data point is an average of three technical replicates. Data shows mean of three independent biological replicates, n=3, Error bars: s.d. e. Western blot analysis of the chromatin-bound fraction of various human 3xFLAG-SLX4 mutants transiently expressed for 48 hours and blotted with anti-FLAG antibody. f and g. Impact of hSLX4 variants on Tus/Ter-induced STGC (f) and I-SceI-induced STGC (g), following transient SLX4 expression in Slx4^+/− and Slx4^ΔUBZ/− Ter-HR reporter clones. Analysis was performed using unpaired Student’s t-test, n=6. Error bars: s.e.m. P-value: **p < 0.01.

Fig. 6.. The XPF nuclease domain is required for Tus/Ter-induced STGC.

a. Schematic of wild type XPF protein and XPF mutant lacking the nuclease domain. b. Strategy for generating Xpf^ΔNuc allele. Red half-arrow heads: genotyping primers. Gel shows PCR products on gDNA from Xpf^+/− and Xpf^ΔNuc/− clones and sequencing chromatogram showing the breakpoints in the in-frame mutant (#6) c-h. Quantification of colony formation of Xpf^+/− vs. Xpf^ΔNuc/− clones in the presence of MMC (c) or Olaparib (d) 5-aza-2′-deoxycytidine (e) Zebularine (f) Hydroxyurea (g) and Camptothecin (h). Data show mean of three biologically independent replicates normalized to untreated samples (n=3). Analysis using unpaired Student’s t-test. P-value: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Error bars: s.d. i. Representative images of metaphase spreads showing chromatid breaks (black arrows) and radial chromosomes (black arrowheads) from indicated Xpf mutant cells, either untreated or treated with 20 ng/mL MMC for 12h. j and k. Quantitation of the number of chromatid breaks (j) radial chromosomes (k) per metaphase in Xpf^+/− (#6) and Xpf^ΔNuc/−(#1) clones. Treated and untreated cells were harvested at the same time and each sample represented one independent experiment, n=1, Analysis using unpaired Student’s t-test. P-value: *p < 0.05; ****p < 0.0001. Error bars: s.d. l and m. Tus/Ter-induced STGC (l) and I-SceI induced STGC (m) in Xpf^+/− vs. Xpf^ΔNuc/− clones. Data shows mean values of six biologically independent replicates, n=6. Analysis by one-way ANOVA. P-value: ****p < 0.0001. Error bars: s.e.m.

Fig. 7.. Model of SLX4 action in Tus/Ter-induced HR.

a. In wild type cells, FA pathway activation and asymmetric fork reversal at forks bidirectionally stalled at Tus/Ter RFB enables SLX4 recruitment and its retention at the stall site, mediated by interactions of the SLX4 UBZ domain with as yet unidentified ubiquitinated components at the stall site. SLX4 MLR domain enables recruitment of XPF-ERCC1, which incises the stalled leading strand of at least one sister chromatid (incision a, red triangle). Note that the second incision (red triangle b), which would be required for ICL unhooking, may be redundant for generation of a DSB at Tus/Ter, since there is no ICL. The two-ended DSB generated by SLX4-XPF is repaired by conservative HR (STGC). b. In cells lacking the SLX4 UBZ domain, retention of SLX4-XPF at the stall site is impaired, leading to inefficient incision of the stalled forks and reduced DSB formation. There is a corresponding defect in Tus/Ter-induced STGC. c. In cells lacking the SLX4 MLR domain, or lacking the XPF nuclease domain, XPF-mediated incision of the stalled forks is abolished, resulting in a severe defect in Tus/Ter-induced STGC.