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

Abstract

Vertebrate replication forks arrested at interstrand DNA crosslinks (ICLs) engage the Fanconi anemia (FA) 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 FANCQ/XPF/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 crosslinking 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.

Introduction

Chromosomal DNA replication is frequently challenged by events that impede progression of the replication fork. Fork-stalling lesions include various types of DNA damage, unusual DNA secondary structures in undamaged DNA, DNA-protein complexes and products of transcription ^1–4. Interstrand crosslinks (ICLs) are formidable fork barriers, since they covalently link the two parental strands, preventing passage of the replicative helicase. Unless the ICL can be disrupted or bypassed, it is an absolute block to replication fork progression ^5–8. In higher eukaryotes, ICL-stalled forks are repaired by the Fanconi anemia (FA) pathway ^9–11. Cells from FA patients are hypersensitive to ICL-inducing agents, including aldehydes, which are an endogenous source of both ICLs and DNA-protein crosslinks ^6,12–15. FA mutant cells reveal elevated frequencies of ICL-induced ‘chromatid-type’ aberrations, reflecting a defect in replication-coupled ICL repair. To date, 22 FA genes have been identified.

The fundamental steps of the FA pathway have been revealed by in vitro studies of ICL-containing plasmids replicating in frog egg extracts ^9,16. Provided the ICL is not bypassed ^7,8, bidirectional fork stalling at the ICL triggers replisome disassembly (‘fork collapse’), orchestrated by the E3 ubiquitin ligase TRAIP ¹⁷. Association of the DNA translocase and scaffolding protein FANCM with the stalled fork helps to recruit the FA core complex, a multi-subunit E3 ubiquitin ligase that monoubiquitinates and activates the FANCD2-FANCI heterodimer ^9,18–20. Monoubiquitinated FANCD2-I is a sliding clamp that encircles dsDNA at the stall site ^21,22; in parallel, asymmetric fork reversal may occur ²³. These steps are required for recruitment of the endonuclease scaffold SLX4/FANCP and the associated endonuclease XPF (xeroderma pigmentosum group F)/FANCQ in complex with ERCC1 (excision repair cross complementation group 1) ^24–27. XPF introduces incisions either side of the ICL on one sister chromatid, generating a two-ended double strand break (DSB) opposite an ‘unhooked’ ICL on the unbroken sister chromatid ^25,28–30. The single stranded (ss)DNA gap opposite the unhooked ICL is filled by translesion synthesis (TLS) DNA polymerases, and the DSB is repaired by conservative HR. Despite these mechanistic insights, it is unclear whether the full FA mechanism is restricted to ICL repair, or whether FA-mediated incisions can induce HR at other types of fork-stalling lesions, such as DNA-protein complexes ³¹. In this regard, a molecularly defined system that recapitulates the FA mechanism in vivo on a mammalian chromosome has been lacking.

To study repair triggered by replication fork stalling at a defined chromosomal locus of cycling mammalian cells, we adapted the Escherichia coli Tus/Ter replication fork barrier (RFB) for use in mammalian cells ³². An array of six 23 bp Ter repeats, when targeted to a specific genomic locus of mouse embryonic stem (mES) cells and bound by the Tus protein, triggers bidirectional fork stalling and HR at the Tus/Ter RFB. Tus/Ter-induced fork stalling also triggers formation of ~10 kb tandem duplications (TDs) in Brca1 mutant cells, recapitulating the TD phenotype of BRCA1 mutant breast and ovarian cancers ^33–35. Thus, although an artificial site-specific DNA-protein fork barrier, the Tus/Ter RFB models the impact of certain endogenous fork-stalling lesions—presumably endogenous DNA-protein barriers—on DNA repair and genomic instability. Conservative HR at Tus/Ter, also called ‘short tract’ gene conversion (STGC), is a non-crossover pathway mediated by early components of the FA pathway and the canonical BRCA-Rad51 HR pathway ^32,33,36,37. In contrast, Tus/Ter-induced ‘long tract’ gene conversion (LTGC)—a replicative outcome related to break-induced replication (BIR)—is independent of BRCA genes, Rad51 and the FA pathway ³². Notably, HR induced at a site-specific DNA-protein fork barrier in Schizosaccharomyces pombe does not proceed via a DSB intermediate ³⁸. Thus, it is important to determine whether Tus/Ter-induced STGC entails an incision step with formation of a DSB intermediate and, if so, how the incision at Tus/Ter is controlled. To address these questions, we have studied the role of SLX4 in Tus/Ter-induced HR.

SLX4/FANCP encodes a scaffolding protein that binds multiple endonuclease complexes in a cell cycle-dependent manner to cleave branched DNA structures ^39–45. In S phase cells, SLX4 interacts with XPF–ERCC1 via the SLX4 MLR (MEI9- Interaction-Like Region) domain, with SLX1 (synthetic lethal of unknown function 1) via the SBD (SLX1-binding) domains; and with MutSβ via the SLX4 N-terminus ⁴⁶. In mitosis, SLX4 additionally binds the MUS81 (methyl methanesulfonate and UV sensitive 81)–EME1 (essential meiotic endonuclease 1) nuclease via the SAP (SAF-A/B, Acinus and PIAS) domain. These interactions enable the SLX4 ‘trinuclease’ complex to act on a variety of branched DNA substrates in mitosis, cleaving stalled replication forks at sites of incomplete DNA synthesis and resolving post-replicative Holliday junctions ^39–43. SLX4 contains at its N-terminus two conserved Ubiquitin Binding Domain 4 (UBZ) motifs that facilitate its localization to laser-induced damage sites, potentially implicating these motifs in a similar recruitment mechanism at stalled forks ^47–49. Notably, all clinically-described FA-associated SLX4/FANCP mutations described to date are hypomorphic alleles, encoding polypeptides with disrupted UBZ motifs ^47,50. More severe SLX4 mutations may be incompatible with development ^51,52.

In this study, we use CRISPR/Cas9 genome editing to engineer precise, defined mutations in the Slx4 gene of mouse embryonic stem (mES) cells that carry a reporter of stalled fork- and DSB-induced HR. We systematically examine the role of individual nuclease-interacting domains of SLX4 in stalled fork HR. Our results identify a unique role for the SLX4-XPF complex in Tus/Ter-induced STGC and reveal a role for the SLX4-XPF in preserving cell viability in response to chemicals that specifically induce DPCs. We define a requirement for the SLX4 UBZ motifs in the recruitment of SLX4 to stalled forks, providing important insight into the mechanisms that connect clinically-described SLX4/FANCP mutations to genomic instability. We also identify an unexpected role for SLX4 in supporting LTGC in the repair of a replication-independent DSB.

Results

Slx4 regulates HR at Tus/Ter-stalled replication forks

To study the role of Slx4 in mammalian stalled fork repair, we used dual CRISPR/Cas9-mediated incisions to introduce defined deletions within the endogenous Slx4 gene of mES cells that contain a single copy of a 6xTer-HR reporter targeted to the Rosa26 locus on chromosome VI ³². In this reporter, HR can be triggered by either Tus/Ter-induced fork stalling or by a site-specific DSB induced by the rare-cutting homing endonuclease I-SceI (Fig. 1a). The reporter enables simultaneous flow cytometric quantitation of error-free HR as short tract gene conversion (STGC; GFP⁺RFP⁻), and aberrant replicative HR responses as long tract gene conversion (LTGC; GFP⁺RFP⁺). We engineered a 125 bp frame-shift deletion in exon 2 (Slx4^Δ125), thereby disrupting Slx4 early in the ORF (Fig. 1b). CRISPR/Cas9 targeting frequently generates a non-synonymous deletion within the second allele of the target gene ^37,53. In recognition of this fact, we term the second allele in Slx4^Δ125 clones “Slx4^Δ”. We assayed two independent Slx4 mutant clones (Slx4^Δ125/Δ #40, 42) and two isogenic clones that received the same CRISPR/Cas9 treatment but retained wild type Slx4 (Slx4^+/+ #3, 13) (Fig. 1b). Both Slx4^Δ125/Δ clones expressed normal levels of Slx4 mRNA (Extended data Fig. 1a), suggesting that Slx4^Δ125 may not be a null allele. Colony formation assays showed that Slx4^Δ125/Δ mutants are hypersensitive to the ICL-inducing agent mitomycin C (MMC; Fig. 1c).

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.

To measure the impact of Slx4 disruption on stalled fork HR and DSB-induced HR, we transfected Slx4^Δ125/Δ Ter-HR reporter cells, in parallel, with Tus or I-SceI expression vectors and assayed repair products 72 hours post-transfection (see methods). Slx4^Δ125/Δ clones revealed a ~4-fold reduction in Tus/Ter induced STGC compared to isogenic wild type clones, whereas Tus/Ter-induced LTGC was unaltered (Fig. 1d, 1e and 1f). Hence, HR at Tus/Ter was skewed in favor of LTGC (Extended data Fig. 1b). In contrast, I-SceI-induced STGC was unaltered in Slx4^Δ125/Δ cells, whereas I-SceI-induced LTGC was reduced (Fig. 1d, 1g and 1h). Consequently, the ratio of I-SceI-induced LTGC:Total HR was reduced in Slx4^Δ125/Δ cells (Extended data Fig. 1c). These results suggest that mammalian Slx4 has distinct roles in stalled fork HR (where it promotes STGC) and in conventional DSB-induced HR (where it mediates LTGC).

To avoid generating non-synonymous Slx4 allelic deletions in subsequent experiments, we established an Slx4 haploid reporter cell line (here termed Slx4^+/−). We used dual CRISPR/Cas9 incisions at exon 2 and exon 15 to delete one copy of the entire 19.3 kb gene (Extended data Fig. 1d, 1e and 1f). We retrieved no Slx4^–/− clones from this experiment, despite an expected frequency of (60/262)² = ~13/262 (Extended data Fig. 1d), suggesting that a true Slx4 null is lethal in mES cells. We used direct sequencing of breakpoint PCR products to identify the deletion breakpoint in Slx4^+/− cells and confirmed that the retained allele is wild type at the sgRNA target sites (Extended data Fig. 1f). Slx4 expression in the Slx4^+/− clone was detected at wild type levels, suggesting that transcription is upregulated in compensation for hemizygosity (Extended data Fig. 1g). Slx4^+/− cells revealed no increased sensitivity to MMC or to the PARP inhibitor Olaparib (Extended data Fig. 1h and 1i) and were phenotypically wild type for stalled fork- and DSB-induced HR (Extended data Fig. 1j).

The SLX4 UBZ domain recruits SLX4 to stalled forks for HR

To study the SLX4 UBZ domain in Tus/Ter-induced HR, we used dual CRISPR/Cas9-mediated incisions to delete in-frame the UBZ1- and UBZ2-coding regions in Slx4^+/− Ter-HR reporter cells (Fig. 2a, Extended data Fig. 2a). UBZ-encoding mRNA sequences were undetectable in Slx4^ΔUBZ/− cells, whereas sequences downstream (encoding MLR, SAP and SBD domains) were expressed at normal levels (Extended data Fig. 2b). Slx4^ΔUBZ/− clones were hypersensitive to MMC (Extended data Fig. 2c), but resistant to Olaparib, Hydroxyurea and Camptothecin (Extended data Fig. 2d, 2g, and 2h; mES cells lacking Brca1 exon 11 served as positive controls in these colony formation assays). Notably, Slx4^ΔUBZ/− cells were hypersensitive to 5-aza-2’deoxycytidine and zebularine—two agents that induce DPCs but not ICLs (Extended data Fig. 2e and 2f) ^54,55. A characteristic feature of FA cells is enhanced S/G2 phase accumulation following exposure to MMC ¹⁰. Untreated Slx4^ΔUBZ/− cells revealed cell cycle distribution patterns similar to Slx4^+/− cells (Fig. 2b). However, in response to titrated doses of MMC, Slx4^ΔUBZ/− cells accumulated in G2/M more readily than Slx4^+/− controls and revealed a corresponding reduction in the S-phase fraction (Fig. 2b).

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.

We analyzed repair functions of six independent Slx4^ΔUBZ/− clones and six independent isogenic Cas9/sgRNA-exposed Slx4^+/− clones. Strikingly, Tus/Ter-induced STGC was reduced ~6-fold in Slx4^ΔUBZ/− clones in comparison to Slx4^+/− controls (Fig. 2c). I-SceI induced STGC was reduced to a lesser extent (Fig. 2d). Transient expression of wild type human SLX4 restored Tus/Ter-induced STGC to wild type levels in Slx4^ΔUBZ/− clones (#2 and #5), but had no impact on Slx4^+/− controls (#25 and #44) (Fig. 2e). A UBZ 4C>A mutant that specifically disrupts UBZ domain function²⁴ failed to complement the STGC defect of Slx4^ΔUBZ/− cells, despite equivalent levels of expression (Extended data Fig. 2i and Fig. 2j). In the same experiment, expression of wild type SLX4 or 4C>A had no statistically significant impact on I-SceI-induced STGC in either Slx4^ΔUBZ/− or Slx4^+/− clones (Fig. 2f). The ability of wild type SLX4 but not the 4C>A mutant to complement the defect in Tus/Ter-induced STGC in Slx4^ΔUBZ/− cells indicates a specific requirement for the SLX4 UBZ domain in error-free HR (i.e., STGC) at stalled forks. In contrast, the failure of wild type SLX4 to complement the modest defect in I-SceI-induced STGC in Slx4^ΔUBZ/− cells provides no clear evidence of a role for the SLX4 UBZ domain in this function. Thus, Slx4^ΔUBZ is a separation-of-function allele that distinguishes control of STGC at stalled forks from STGC at replication-independent DSBs.

The same experiments revealed different effects on LTGC. Tus/Ter-induced LTGC was modestly reduced in the six Slx4^ΔUBZ/− clones; hence, the ratio of LTGC:Total HR was elevated, reflecting the severe defect in STGC (Extended data Figs. 3a and 3b). Tus/Ter-induced LTGC was unaffected by expression of either wild type SLX4 or the 4C>A mutant (Extended data Fig. 3c). Thus, there is no clear evidence of a role for the SLX4 UBZ domain in Tus/Ter-induced LTGC. In contrast, I-SceI-induced LTGC was reduced >2-fold in the six Slx4^ΔUBZ/− clones in comparison to Slx4^+/− controls (Extended data Fig. 3d); as a result, the ratio of LTGC:Total HR was reduced ~2-fold, reflecting the specific reduction in I-SceI-induced LTGC (Extended data Fig. 3e). Importantly, the LTGC defect was complemented by wild type SLX4 but not by the 4C>A mutant (Extended data Fig. 3f). Thus, Slx4^ΔUBZ is a separation-of-function allele that distinguishes control of LTGC at replication-independent DSBs from LTGC at stalled forks.

To facilitate detection of the endogenous Slx4 gene product in mES cells, we targeted the 3’end of the residual Slx4 allele in Slx4^+/− and Slx4^ΔUBZ/− cells with a dual minimal auxin-inducible and SMASh degron containing an 8x HA epitope tag positioned N-terminal of the SMASh degron cleavage site ^37,56–58, to generate Slx4^deg/− and Slx4^ΔUBZdeg/− clones (Fig. 2g). Slx4^deg/− and Slx4^ΔUBZdeg/− clones expressed comparable levels of Slx4 mRNA (Fig. 2h). We detected non-specific bands in the whole-cell extract (WCE), however, a specific HA-tagged gene products were detectable in the chromatin fraction (Extended data Fig. 2k, Fig. 2i). Therefore, we restricted our western blot analyses to chromatin bound Slx4. Activation of the dual degron triggered partial degradation of endogenous SLX4, confirming the specificity of the anti-HA western blot signal. Unexpectedly, the HA-tagged Slx4^ΔUBZdeg gene product was more abundant in the chromatin fraction than the wild type protein (Fig. 2i). We used these tools, in combination with chromatin immunoprecipitation (ChIP), to study SLX4 recruitment to Tus/Ter-stalled forks. Strikingly, we noted robust accumulation of wild type SLX4-deg at the Tus/Ter RFB, but reduced accumulation of SLX4-ΔUBZdeg (Fig. 2j). Thus, the SLX4 UBZ domain mediates Tus/Ter-induced STGC by recruiting SLX4 to the site of fork stalling.

SLX4-MUS81 and SLX1 complexes: no role in Tus/Ter-induced HR

To study the SLX4-MUS81 interaction in Tus/Ter-induced repair, we used Cas9-dual sgRNA incisions to generate in-frame deletions of Slx4 exons 11–13, encoding the SAP domain, in Slx4^+/− Ter-HR reporter cells (Fig. 3a and Extended data Fig. 4a). The mRNA sequence encoding the SAP domain was undetectable in Slx4^ΔSAP/− cells, whereas expression of other Slx4 elements was at wild type levels (Extended data Fig. 4b). Degron-HA-targeted Slx4^ΔSAP/− cells revealed normal levels of HA-tagged gene products (Extended data Fig. 4c). Slx4^ΔSAP/− cells were modestly sensitive to MMC but not to Olaparib, 5-aza-2′-deoxycytidine, zebularine, hydroxyurea and camptothecin (Extended data Fig. 5a, 5b, 5c, 5d, 5e and 5f) in comparison to isogenic Slx4^+/− cells that had been similarly treated with Cas9/sgRNA. We analyzed stalled fork and DSB repair functions in five independent Slx4^ΔSAP/− clones and five isogenic Slx4^+/− clones and found no impact of SAP domain deletion on any of the repair functions measured (Fig. 3b and 3c; Extended data Fig. 4d and 4e). Thus, the interaction of SLX4 with MUS81 is dispensable for Tus/Ter-induced and I-SceI-induced HR.

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.

To study the SLX4-SLX1 interaction in Tus/Ter induced repair, we used Cas9-dual sgRNA incisions to generate in-frame deletions of Slx4 exons 13–15, encoding the SBD domain, in Slx4^+/− Ter-HR reporter cells (Fig. 3d and Extended data Fig. 4f). The mRNA sequence encoding the SBD domain was almost undetectable in the Slx4^ΔSBD/− cells, whereas expression of other elements of Slx4 was at normal levels (Extended data Fig. 4g). Degron-HA-targeted Slx4^ΔSBD/− cells revealed normal levels of HA-tagged gene products (Extended data Fig. 4h). Slx4^ΔSBD/− cells were insensitive to MMC, Olaparib, 5-aza-2′-deoxycytidine, zebularine, hydroxyurea and camptothecin (Extended data Fig. 5g, 5h, 5i, 5j, 5k and 5l) in comparison to isogenic Slx4^+/− cells that had been similarly treated with Cas9/sgRNA. We analyzed stalled fork and DSB repair functions in six independent Slx4^ΔSBD/− clones and six isogenic Slx4^+/− clones and found no impact of SBD domain deletion on any of the repair functions measured (Fig. 3e and 3f; Extended data Fig. 4i and 4j). Thus, the interaction of SLX4 with SLX1 is dispensable for Tus/Ter-induced and I-SceI-induced HR.

SLX4-XPF binding is required for Tus/Ter-induced STGC

To study the SLX4-XPF interaction in Tus/Ter stalled fork repair, we used dual sgRNAs to generate in-frame deletion of the MLR-encoding exons 5 and 6 in Slx4^+/− Ter-HR reporter cells (Fig. 4a, Extended data Fig. 6a). Slx4 mRNA transcripts in Slx4^ΔMLR/− clones lacked the deleted region but were otherwise detected at normal levels (Extended data Fig. 6b). Degron-HA-targeted Slx4^ΔMLR/− cells revealed a modest reduction in the levels of HA-tagged gene products (Extended data Fig. 6c). Slx4^ΔMLR/− clones were slow-growing compared to isogenic Slx4^+/− controls that had been similarly exposed to Cas9/sgRNA but had retained wild type Slx4. In untreated cells, the proportion of cells in late S/G2/M was elevated ~2-fold in Slx4^ΔMLR/− cells in comparison to Slx4^+/− cells (Fig. 4b). The S-phase fraction was correspondingly reduced. Similarly, following exposure of cells to titrated doses of MMC, Slx4^ΔMLR/− cells revealed more dramatic late S/G2/M accumulation and S phase depletion than Slx4^+/− controls (Fig. 4b). Slx4^ΔMLR/− cells were hypersensitive to MMC, 5-aza-2′-deoxycytidine and zebularine but not to Olaparib, hydroxyurea and camptothecin in comparison to Slx4^+/− controls (Extended data Fig. 6d, 6e, 6f, 6g, 6h and 6i). The slow growth and late S/G2/M accumulation suggest that Slx4^ΔMLR/− cells do not resolve replication stress efficiently. To explore this effect further, we analyzed metaphase spreads from Slx4^+/−, Slx4^ΔUBZ/− and Slx4^ΔMLR/− cultures that were either untreated or exposed to 20 ng/ml of MMC for 12 hours. Both Slx4^ΔUBZ/− and Slx4^ΔMLR/− cells revealed elevated levels of breaks and radial chromosomes in comparison to Slx4^+/− controls, but Slx4^ΔMLR/− cells revealed a more severe defect than Slx4^ΔUBZ/− cells (Fig. 4c, 4d and 4e). Thus, both Slx4^ΔUBZ/− and Slx4^ΔMLR/− cells display a classical Fanconi anemia chromosome breakage/aberration phenotype. In a second experiment, we additionally analyzed Slx4^ΔSAP/− and Slx4^ΔSBD/− cells (Extended data Fig. 6j, 6k and 6l). Slx4^ΔSAP/− cells were indistinguishable from Slx4^+/− cells. Slx4^ΔSBD/− cells revealed modestly elevated frequencies of breaks and radials compared to Slx4^+/− cells.

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.

We analyzed stalled fork- and DSB-induced HR in six independent Slx4^ΔMLR/− clones and six independent Slx4^+/− isogenic clones. Tus/Ter-induced STGC was almost undetectable in Slx4^ΔMLR/− cells, whereas LTGC was detected at wild type levels (Fig. 5a and Extended data Fig. 7a). I-SceI induced STGC was reduced in Slx4^ΔMLR/− clones, while I-SceI induced LTGC was marginally reduced (Fig. 5b and Extended data Fig. 7b). A caveat of these results is the possibility that the slow growth of Slx4^ΔMLR/− cells might artifactually alter the measured HR frequencies ⁵⁹. To address this concern, we performed complementation experiments in Slx4^ΔUBZ/− Ter-HR reporter cells, which exhibit no proliferation defect. In addition to testing full-length hSLX4 expression vectors, we generated ‘mini-SLX4’ constructs that encode hSLX4 polypeptides that are truncated C-terminal to the BTB domain and therefore lack the MUS81-binding SAP or SLX1-binding SBD domains ²⁸. These mini-SLX4 constructs included wild type, 4C>A and ΔMLR variants (Fig. 5c). Following transient transfection, hSLX4 was expressed equivalently from each construct, although the wild type mini-SLX4 protein was more abundant than other products (Fig. 5d and 5e). Expression of mini-SLX4 complemented the defect in Tus/Ter-induced STGC in Slx4^ΔUBZ/− cells as efficiently as wild type SLX4 (Fig. 5f). Importantly, both full length SLX4ΔMLR and mini-SLX4ΔMLR failed to complement the defect in Tus/Ter-induced STGC in Slx4^ΔUBZ/− cells, behaving similarly to mini-SLX44C>A. No significant effects of any of these constructs were observed on Tus/Ter-induced STGC in Slx4^+/− cells, and I-SceI-induced STGC was unaffected by any SLX4 construct in either cell type (Fig. 5f and 5g). These findings show that intact SLX4 UBZ and MLR domains are independently required for Tus/Ter-induced STGC. The absence of interallelic complementation between endogenous Slx4^ΔUBZ and exogenous SLX4ΔMLR alleles suggests that intact UBZ and MLR domains must each be present on the same SLX4 molecule in order for SLX4 to mediate Tus/Ter-induced STGC.

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.

In the same experiments, Tus/Ter-induced LTGC was unaffected by any of the above-noted hSLX4 constructs in either Slx4^ΔUBZ/− or Slx4^+/− cells (Extended data Fig. 7c). In contrast, I-SceI-induced LTGC, which is specifically impaired in Slx4^ΔUBZ/− cells, was complemented by expression of full-length wild type SLX4 (consistent with our previous experiments), but not by the ΔMLR mutant (Extended data Fig. 7d). Similarly, wild type mini-SLX4 complemented the defect in I-SceI-induced LTGC, whereas mini-SLX4 mutants 4C>A and ΔMLR failed to do so. These results suggest that I-SceI-induced LTGC requires the same elements of SLX4 that are required for Tus/Ter-induced STGC.

The XPF nuclease domain in Tus/Ter- and I-SceI-induced STGC

The above findings implicate the SLX4-XPF interaction in Tus/Ter-induced STGC. We therefore established tools to study XPF in stalled fork repair. We used CRISPR/Cas9 with dual sgRNA targeting to delete one entire ~42.3 kb Xpf allele in Ter-HR reporter cells (Extended data Fig. 8a and 8b). Xpf expression was reduced by ~50% in Xpf^+/− cells compared to an isogenic Xpf^+/+ clone, implying an absence of transcriptional compensation for hemizygosity (Extended data Fig. 8c). Nonetheless, Xpf^+/− Ter-HR reporter cells revealed wild type levels of the XPF binding partner ERCC1, MMC and Olaparib resistance, and repair frequencies (Extended data Fig. 8d, 8e, 8f and 8g). (We were unable to detect the endogenous XPF protein in mES cells.) To determine whether the XPF nuclease domain contributes to stalled fork HR, we used Cas9 with dual sgRNAs to delete in-frame the nuclease domain-encoding region of Xpf in Xpf^+/− cells (Fig. 6a and 6b). We obtained one Xpf^Nuc/− clone containing an in-frame deletion (#1) and three that contained a frameshift (#2, #11, #13). Analysis of Xpf expression in each mutant clone revealed the absence of nuclease domain-encoding sequences but normal levels of expression of regions encoding the upstream helicase domain and the downstream HhH2 domain (Extended data Fig. 8h). Western blot analysis of the HhH2-binding XPF partner ERCC1 in whole cell extracts revealed variably reduced abundance of ERCC1 in Xpf^Nuc/−clones (Extended data Fig. 8i). Chromatin association of ERCC1 was detected in Xpf^Nuc/−clone #1 but not in the three Xpf^Nuc/− frameshift clones, suggesting that interaction of XPF with ERCC1 is required for chromatin association of ERCC1 (Extended data Fig. 8j). All four Xpf^Nuc/− clones displayed a proliferative defect similar to that of Slx4^ΔMLR/− cells, associated with in an increase in the G2/M fraction and a reduction in the S-phase fraction in unperturbed cells (Extended data Fig. 8k). Similarly, following exposure of cells to titrated doses of MMC, Xpf^Nuc/− cells revealed more dramatic G2/M arrest and S phase depletion than Xpf^+/− controls. Xpf^Nuc/− cells were hypersensitive to MMC, 5-aza-2’deoxycytidine and zebularine, and modestly sensitive to Olaparib and camptothecin, in comparison to Xpf^+/− controls (Fig. 6c, 6d, 6e, 6f and 6h). Xpf^Nuc/− cells were resistant to hydroxyurea treatment (Fig. 6g). We prepared metaphase spreads from Xpf^Nuc/− or Xpf^+/− cells that were either untreated or exposed to 20 ng/ml MMC. Xpf^Nuc/− cells revealed dramatically elevated frequencies of MMC-induced chromatid breaks and radials, consistent with the status of XPF/FANCQ as a FA gene (Fig. 6i, 6j and 6k).

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.

We analyzed stalled fork and DSB repair functions in each of the four Xpf^Nuc/− Ter-HR reporter clones and four isogenic Xpf ^+/− clones. Similar to Slx4^ΔMLR/− cells, Xpf^Nuc/− cells revealed near-complete loss of Tus/Ter-induced STGC (Fig. 6l) but no alteration in Tus/Ter induced LTGC (Extended data Fig. 8l). We also noted a significant reduction in I-SceI induced STGC (Fig. 6m) and a modest reduction in I-SceI induced LTGC (Extended data Fig. 8m). Thus, Xpf^Nuc/− cells phenocopy Slx4^ΔMLR/− cells in stalled fork repair. The slow growth phenotype of Xpf^Nuc/− cells suggests that these repair frequencies should be interpreted with caution. However, all of our experiments point to SLX4-XPF as a critical and specific mediator of Tus/Ter-induced STGC. Further, the Slx4 and Xpf mutants with the most severe FA phenotype exhibited the most profound impairment of Tus/Ter-induced STGC and were hypersensitive to DPC-inducing agents.

Discussion

We show here that the structure-specific endonuclease scaffold SLX4 has a critical and specific function in conservative HR (i.e., STGC) at Tus/Ter-stalled forks. SLX4 ubiquitin-binding UBZ and XPF-binding MLR domains are required for Tus/Ter-induced STGC, while the SAP and SBD domains are dispensable, excluding a requirement for the mitosis-associated SLX4 trinuclease complex. Taken together, our findings suggest that a major function of SLX4 in Tus/Ter-induced STGC is to position XPF at the stalled fork, promoting XPF-mediated incision of the bidirectionally Tus/Ter-arrested fork (Fig. 7). Such a function would recapitulate the critical role of SLX4-XPF in the ‘unhooking’ step of ICL repair ^9,25,30, with the exception that only one XPF-mediated incision would be required to generate a two-ended DSB at Tus/Ter because of the absence of a crosslink. This model explains why SLX4 plays a major role in Tus/Ter-induced STGC but not in I-SceI-induced STGC. In the former case, if SLX4-XPF coordination at the stalled fork were defective, production of a DSB at the stall site would be suppressed, resulting in low levels of HR. In the latter case, the DSB is induced by the I-SceI endonuclease, bypassing any requirement for SLX4-XPF in DSB formation. SLX4-XPF might have additional generic roles in HR, such as in flap-processing.

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.

Key components of the FA pathway required for efficient Tus/Ter-induced STGC include FANCM ³⁷, the FA core complex ^33,37, the BRCA-Rad51 pathway ⁶⁰ and SLX4-XPF (this study). In light of these findings, it will be important in future to determine whether Tus/Ter-induced STGC requires TRAIP-mediated CMG ubiquitination ¹⁷, monoubiquitination of FANCD2-FANCI ²⁵ and/or a fork reversal step ²³. If, indeed, the full FA mechanism is required for Tus/Ter-induced STGC, this would extend the scope of the FA pathway beyond ICL repair and indicate that the FA pathway can also process some DNA-protein replication fork barriers for HR ³¹. In support of this idea, we find that cells defective for SLX4-XPF are hypersensitive to 5-aza-2’-deoxycytidine and zebularine. These cytidine analogs are incorporated into DNA and trap de novo DNA methyltransferases (DNMTs) in chromatin as covalent complexes (i.e., DPCs), but do not form ICLs ^54,55. Importantly, the cellular toxicity of 5-aza-2’-deoxycytidine is caused by its capacity to cross-link DNMTs to DNA, not by downstream effects on DNA methylation ⁵⁴. Although it has been shown that the FA pathway is not necessary for repair of one class of DPCs, some DPCs encountered during replication might escape proteolytic mechanisms of DPC bypass and be processed by the FA pathway for HR ^31,61–64. Ongoing experiments are underway to determine whether FA pathway components in addition to SLX4 and XPF are involved in 5-aza-2’-deoxycytidine resistance. In this regard, aldehydes, a class of chemicals to which FA mutant cells are hypersensitive, are known to form both ICLs and DPCs ^6,12–15. The Tus/Ter system may prove to be a useful model of FA-mediated repair of endogenous DPCs.

We show that a key function of the SLX4 UBZ domain is to support efficient retention of SLX4 at the stall site. As noted above, specific inactivation of UBZ domain function is a common defect of clinically-described FANCP mutations ^47,50. Our data suggest that a key defect in FANCP mutant FA patient cells is reduced retention of SLX4 at stalled forks. Unlike the true SLX4 null, which may be cell lethal when homozygous ^51,52 (and this study), germ line homozygous SLX4 ΔUBZ mutations may be compatible with human development because the mutation is hypomorphic—reducing, but not abolishing, SLX4 action at stalled forks.

Unexpectedly, we identified SLX4 as a mediator of DSB-induced LTGC. This finding might appear to connect DSB-induced LTGC to mitotic DNA synthesis (MiDAS), which is mediated by SLX4 ⁶⁵, MUS81 ⁶⁶ and SLX1 ⁶⁷. However, interaction of SLX4 with MUS81 or SLX1 is dispensable for DSB-induced LTGC, i.e., there is no indication of a role for the mitotic SLX4 trinuclease complex. Instead, DSB-induced LTGC requires the SLX4 UBZ and MLR domains, implicating ubiquitin chain binding and XPF in the mechanism. Further studies will be needed to decipher how SLX4-XPF-mediated incisions contribute specifically to DSB-induced LTGC.

Methods

Molecular biology and sgRNA oligos

The 6xTer-HR reporters used were assembled using standard cloning methods described previously for the 6xTer-HR reporter ^32,33. Derivation of Tus and I-SceI expression plasmid are described in ^32,68. Ter-containing plasmids were amplified in JJC33 (Tus⁻) strains of E. coli. All plasmids used for transfection were prepared by endotoxin-free maxiprep (QIAGEN Sciences). All primers used for genotyping, RT-qPCR, sgRNA synthesis, and chromatin immunoprecipitation were purchased from Life Technologies.

Generation of mouse Slx4 and Xpf mutant cell lines and cell culture

Slx4 mutant cell lines were derived from a conditional mouse Brca1^fl/exon11 Ter-HR reporter ES cell line ³². This founder cell line contains a single copy of the 6x Ter-HR reporter cassette, targeted to the Rosa26 locus and verified by southern blot. Mouse ES cells are routinely thawed onto plates coated with mouse embryonic fibroblast (MEF) feeders, maintained in ES medium on gelatinized plates, and regularly tested for mycoplasma infection by Myco-Alert assay (Lonza). Slx4 mutant cell lines were generated using CRISPR/Cas9 mediated mutation of the Slx4 locus. The genotype of each cell line was defined directly using specific PCR testing and cell lines were re-genotyped on multiple occasions. sgRNAs targeting Cas9 to the Slx4 locus were transcribed in vitro using the Engen sgRNA Synthesis kit (New England Biolabs E3322S), purified using the RNA Clean and Concentrator Kit (Zymo Research, R1017), and verified by denaturing 10% TBE-urea PAGE. Cas9-sgRNA RNP was pre-assembled in vitro in OptiMEM by mixing Spy NLS Cas9 (New England Biolabs, M0646T) and purified sgRNA. Cells were co-transfected with either 0.45 μg Cas9(1.1) expression plasmid and 0.05 μg of each purified sgRNA using Lipofectamine 2000 (Invitrogen). After 72 hr, transfected cells were plated onto 6-cm dishes containing feeder MEFs without selection. Individual clones were picked for expansion between 9 and 14 days later and Slx4 mutant clones were identified by PCR and confirmed by sequencing. Slx4 targeting sgRNA sequences including PAM: Slx4^Δ125/Δ 5’ exon 2 (5’ CCT TCC TGT TTC ACG ACA ACT GG 3’), Slx4^Δ125/Δ 3’ exon 2 (5’ ATC CCC CCA AAA GAC TGC ACT GG 3’), Slx4 hemizygote exon 2 (5’ ATC CCC CCA AAA GAC TGC ACT GG 3’), Slx4 hemizygote exon 15 (5’ ATC CTT TTT CTT TCG GC CTG AGG 3’); Slx4 ΔUBZ 5’ (5’ ATG ACA GCC TGG AGG AGA AGG GG 3’), Slx4 ΔUBZ 3’ (5’ ACA GCC TGA AGC AGG AGT TGG GG 3’); Slx4 ΔMLR 5’ (5’ CCA AAA GGG AGC CAA GGA GGA GG 3’), Slx4 ΔMLR 3’ (5’ GGG CAT TGG GAC ACA ATT GGA GG 3’); Slx4 ΔSAP 5’ (5’ CCA GAA CTC GGA AGC CAG ACA GG 3’), Slx4 ΔSAP 3’ (5’ GGT AGT TCT GCG GGT ATC TGG GG 3’); Slx4 ΔSBD 5’ (5’ CGC AGA ACT ACC TTG TCG CCA GG 3’), Slx4 ΔSBD 3’ (5’ GAA AAA GGA TCA GAA ATG ACA GG 3’); sgRNA targeting Slx4 dual degron (5’ ATC CTT TTT CTT TCG GCC TGA GG 3’); Xpf hemizygote 5’ UTR (5’ GTG CGG CCG CGG TCC GAA GGG 3’), Xpf hemizygote exon 11 (5’ CAA ACA GCT CGG CTG TGG CGT GG 3’); Xpf ^Nuc/− 5’ (5’ CTC TGC CTT CCC GTT CTT CGG GG 3’), Xpf ^Nuc/− 3’ (5’ ACC AGA GCA GCC GCA AAC GCG GG 3’).

Recombination assays

These assays were performed as described in detail in ⁶⁹.Briefly, 1.6 × 10⁵ cells were co-transfected in suspension with 0.5 mg empty vector, pcDNA3b-myc NLS-Tus, or pcDNA3b-myc NLS-I-SceI using Lipofectamine 2000 (Invitrogen). GFP+RFP–, GFP+RFP+ and GFP–RFP+ frequencies were scored 72 hours after transfection by flow cytometry Beckman Coulter CytoFlex LX. For each duplicate sample condition, 3–6 × 10⁵ total events were scored. Repair frequencies presented are corrected for background events and for transfection efficiency (50–85%). Transfection efficiency was measured by parallel transfection with 0.05 mg wild type GFP expression vector and 0.45 mg empty vector. For transient hSLX4 rescue experiments, 1.6 × 10⁵ cells were co-transfected in suspension with 0.4 mg empty vector, pcDNA3b-myc NLS-Tus ³², or pcDNA3b-myc NLS-I-SceI ⁶⁸, and either 0.1 mg empty vector and pHIV- 3xFLAG-AttB1-hSlx4-AttB2- NatR-T2A-hCD52 and all other Slx4 domain mutant derivatives using Lipofectamine 2000.

Colony formation assay

Approximately 2000 ES cells were plated on 6 cm tissue culture dishes coated with 0.2% gelatin. 18–20 hours after plating, MMC, Olaparib, 5-aza-2′-deoxycytidine or Zebularine was added to the media. After 7–10 days, cells were fixed using Carnoy’s fixative (75% methanol + 25% glacial acetic acid) and stained with crystal violet. Colonies were counted using a Celigo machine. Graphs represent the mean of duplicate samples from three independent experiments. The experiments were blinded during colony counting. Note: all colony formation assays were performed as a single experiment, with subsets of treatment groups shown in individual figures. Brca1 exon 11 mutant mES cells served as positive controls for sensitivity to MMC and olaparib. This control is shown in Supplementary Fig. 2c-2f.

RT-qPCR

RNA extraction was performed using QIAGEN RNeasy Mini Kit (QIAGEN Sciences,). All analyses of Gapdh and genes of interest were performed using an Applied Biosystems 7300 Real time PCR System using Power SYBR Green RNA-to CT ^™ 1-Step Kit (Applied Biosystems, Foster City, CA).Primers for RT-PCR: Gapdh sense (5’ CGT CCC GTA GAC AAA ATG GT 3’); Gapdh antisense (5’ TCG TTG ATG GCA ACA ATC TC 3’); Slx4 exon2 frameshift sense (5’ CAG AGA CAC CAA AAG GTG CTA ACC 3); Slx4 exon2 frameshift antisense (5’ GCA CGG ACT CCA AAC CTG TCC AGT 3’); Slx4 hemizygote sense (5’ CAG AGA CAC CAA AAG GTG CTA ACC 3’); Slx4 hemizygote antisense (5’ GCA CGG ACT CCA AAC CTG TCC AGT 3’); Slx4-UBZ sense (5’ GCA ACA TGT GAA CAG ATG CCT); Slx4-UBZ antisense (5’ TTG GTG GTG AGG AAC GGT TT 3’); Slx4-MLR sense (5’ ACC ACC AAA AGG GAG CCA AG 3’); Slx4-MLR antisense (5’ CTT GCG ACT TTT CTT CTC TGC T 3’); Slx4-SAP sense (5’ CAG AGA CAC CAA AAG GTG CTA ACC 3’); Slx4-SAP antisense (5’ GCA CGG ACT CCA AAC CTG TCC AGT 3’); Slx4-SBD sense (5’ CTG CAA CCC TTC AAG GTT GCC AAC 3’); Slx4-SBD antisense (5’ GAG GAG CTT TTA GAG CTA AAG GAG 3’), Xpf hemizygote sense (5’ GGG AAA CCT CTG AGG GTT TAC TTT 3’), Xpf hemizygote antisense (5’ CTA GCT TTT TCT CTT ATG AGC TTT 3’), Xpf helicase sense (5’ GTC AGA ATT CAG GTT GGC TTT TCC 3’); Xpf helicase antisense-(5’ TTG TTT CTT GAA CTT CTG GAC TCG 3’), Xpf nuclease sense (5’ GAA AAG CTC ATA AGA GAA AAA GCT 3’); Xpf nuclease antisense-(5’ CTC CTG GCC ACC GGC TTT CCG AGT 3’); Xpf Hhh2 sense (5’ GAA GAT GCC TGG GGT CAA CGC CAA 3’); Xpf Hhh2 antisense-( 5’ AGG AAG TCG TGC AGC TGC TTG GCA T 3’). N-terminal FLAG tagged human SLX4 sense (5’ ATG TGA ACA GGT GCT TGG ATG AAG 3’), antisense (5’ ATC ACT GAA GCT GAA CAT GGG TGG 3’). mRNA levels were measured in technical triplicates. Target gene expression level was normalized to Gapdh using the 2^–ΔCT method and the mutants were normalized to the corresponding wild-type.

Isolation of Chromatin-bound Slx4 and ERCC1 western blot

~10×10⁶ cells were freshly harvested and washed with 1x PBS and lysed in 5 volumes of chilled A1 buffer (50mM HEPES,140mM Nacl,1mMEDTA,10%glycerol,0.5% NP-40,0.25% TritonX-100,1mM DTT, and 1x protease inhibitor). Each lysate was centrifuged at 1,100 x g at 4 °C for 2 min and the supernatant was discarded. Pellets were resuspended by gentle pipetting in A1 buffer and samples were incubated for 10 minutes on ice. Each suspension was centrifuged at 1,100 x g at 4 °C for 2 min and the supernatant again discarded. Pellets were resuspended by gentle pipetting in 2 volumes of ice cold E2 buffer (10mM Tris-HCl pH-8,200mMNacl, 1mM EDTA and 0.5mM EGTA, and 1x Protease inhibitor). Each suspension was centrifuged at 1,100 x g at 4 °C for 2 min and the supernatant discarded. Pellets were resuspended by gentle pipetting with A1 buffer and incubated for 10 minutes on ice. Each suspension was centrifuged at 1,100 x g at 4 °C for 2 min and the supernatant discarded. Pellets were resuspended by gentle pipetting in 2 volumes of ice cold E3 buffer (500mM Tris-HCl pH-6.8, 500mMNacl and 1x Protease inhibitor. Each sample was sonicated in a water bath using Diagenode Bioruptor 300 with attached 4°C chiller cycling 30 sec on and 30 sec off on high setting for 5 min. Sonicated samples were centrifuged at 16,000xg for 15 min at 4°C. The supernatant corresponding to the chromatin fraction was transferred and subjected for western blot analysis using following antibodies; anti-HA (Abcam ab9110, 1:1000), anti-ERCC1 (Santacruz sc-17809, 1:250), Histone H3 (Abcam ab1791, 1:3000).

Chromatin immunoprecipitation Assay

Chromatin immunoprecipitation was performed as described previously ⁷⁰. Transfection of 2.0 × 10⁵ Ter-HR reporter cells of the genotypes noted was performed with 0.5 μg empty vector, pcDNA3β-MYC NLS-I-SceI or pcDNA3β-MYC NLS-Tus-F140A-3xFlag using Lipofectamine 2000 (Invitrogen). 10 million cells were collected 24 hours after transfection and fixed in 1% formaldehyde supplemented serum free media. Fixation was quenched by addition of glycine to 125 mM. Cells were lysed in lysis buffer (0.1% SDS, 20 mM EDTA, 50 mM Tris pH 8.1) containing protease inhibitor (Roche protease inhibitor, Roche 13539320). Chromatin shearing to ~500 bp was accomplished using Diagenode Bioruptor 300 with attached 4°C chiller for 20 cycles, 15 seconds on and 30 seconds off at medium setting. To avoid non-specific binding to protein A/G beads, 100 μl lysates for each ChIP sample were precleared by the addition of 10 μl activated Magna ChIP magnetic beads (Millipore Sigma, 16–663) in ChIP dilution buffer (1% Triton-X-100, 2mM EDTA, 150mM NaCl, 20mM Tris pH 8.1) and incubation for 1 hr at 4°C with gentle mixing. After removal of beads by magnet, for each immunoprecipitation, 7 μl of anti-HA (Abcam ab9110) was added and mixed for 12 hours at 4°C followed by addition of 10 μl activated Magna ChIP beads and mixing for 16 hours at 4°C. Beads were washed six times in ice-cold ChIP RIPA buffer (50mM HEPES pH 7.6, 1mM EDTA, 7 mg/mL sodium deoxycholate, 1% NP-40) followed by two washes in ice-cold TE (10 mM Tris pH8.0, 1 mM EDTA). Crosslinks were reversed and DNA was eluted by incubation in 100 μL Elution buffer (1% SDS, 200mM sodium bicarbonate, 5.6 μg/mL RNAse A) overnight at 65°C. Protein was removed by proteinase K digest for 30 min at 55°C. Released DNA was purified by Qiagen PCR Purification column (Qiagen, 28106) and analyzed by qPCR on an ABI Prism 7300 or QuantStudio3 using SYBR Green (Applied Biosystems, 4368702). Primers for qPCR: +109 bp sense (TCC GGA TAG GGA TAA CAG GGT A); +109 bp antisense (GTC GGC CAT GAT ATA GAC GTT G); 128 bp sense (GAG CGC ACC ATC TTC TTC A); 128 bp antisense (TCC CTA CGA TGC CCT TCA). Data are presented as the mean calculated from three independent experiments (n = 3) normalized against untreated controls (empty vector) and control locus (beta-actin) using the 2^–ΔΔCT method ⁷¹.

Cell Cycle Analysis

ES cells were grown on a 6-well plate until ~70% confluent. Cells were treated with varying concentrations of MMC for 12 h and compared with untreated controls. After 12h, cells were pulsed with 10 μM BrdU for 15 min. Harvested cells were then washed with pre-chilled 1x PBS pH 7.0 by gently pipetting it and spun at 1000 rpm for 5 mins. Supernatant was discarded and the pellet was gently resuspended in 50μl PBS. Cells were fixed by adding 5mL of chilled 70% Ethanol in a dropwise manner while gently but constantly vortexing to prevent clumping. Fixed cells are stored at 4°C in the dark. Before immunostaining for BrdU, cells were spun at 1000rpm for 5 mins to remove the ethanol. While gently vortexing, 0.5mL of 2M HCl, 0.5% Triton-X-100 solution was added in a dropwise manner to denature DNA. Cells were incubated at RT for 30 mins, with intermittently vortexing approximately every 10 mins. Excess acid was removed by spinning and cells were neutralized using 1mL 0.1M Sodium Borate Decahydrate. Once the neutralizing solution is removed, resuspend pellet in 50–100μl of 1xPBS containing 1%BSA. BrdU was counterstained using anti-BrdU antibody (Abcam ab8039, 1:100) and cells were incubated at RT for 30 mins in the dark. Cells were then washed with 5mL chilled 1xPBS containing 20mM HEPES pH 7.4, 0.5% tween-20 to remove excess antibody. A secondary FITC-conjugated rabbit anti-mouse antibody (Jackson Immunoresearch, 115–095-146–1:50) was used and cells were incubated at RT for 30 mins in the dark. Cells were then washed with 5mL chilled 1x PBS containing 20mM HEPES pH 7.4, 0.5% tween-20 to remove secondary antibody. Cells were resuspended with 300μl of solution containing 38mM sodium citrate, 69μM propidium iodide, 5μl/mL RNaseA in the dark for 15 mins at 37°C. Approximately 10⁴ events were acquired using Cytoflex LX and the results were analyzed using FloJo software.

Chromosomal breakage assay

~ 3×10⁶ adherent cells were either untreated or treated with 0.02μg/mL MMC for 12h. Cells were arrested with colcemid (0.17 μg per ml of media) for 20 min. Cells were harvested and incubated at 37°C in 0.075 M KCl for 10 mins and fixed in freshly prepared methanol:glacial acidic acid (3:1 vol/vol). Cells were stored at 4°C at least overnight. Cells were dropped onto wet slides and air dried at 40°C for 60 min before staining with KaryoMAX Giemsa (Invitrogen) Gurr Buffer for 3 min. After rinsing with fresh Gurr Buffer followed by distilled water, the slides were fully dried at 40 °C for 60 min and scanned using the Metasystems Metafer application. The experiments were randomized and breaks, and radial chromosomes were scored by a person blinded to the identity of the samples.

Statistical methods

Data shown represents the arithmetic mean and error bars represent either the standard error of the mean (s.e.m.) or standard deviation (s.d.) of between three (n = 3) and nine (n = 9) independent experiments (as noted in each Figure Legend). Data points for each independent experiment were typically collected as the mean of technical duplicates. This mean value was taken as the solitary data point for that individual experiment. The arithmetic mean of samples collected for groups of independent experiments for repair frequency statistical analysis, was calculated and data points for each independent experiment used to calculate the mean and s.e.m., calculated as standard deviation/√ n, (n indicates the number of independent experiments). Differences between sample pairs repair frequencies and fold enrichment for ChIP were analyzed by Student’s two-tailed unpaired t-test, assuming unequal variance. One-way ANOVA statistical analysis of greater than three samples was performed when indicated. P-values are indicated in the figure and the P-value code for each figure is as written in the Fig. 1 legend. No statistical methods were used to predetermine sample size. The experiments were not randomized, and investigators were not blinded to allocation during experiments and outcome assessment. All statistics were performed using GraphPad Prism v7.0d software.

Extended Data

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.

Data Availability

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