Molecular basis of XPF-ERCC1 targeting to SLX4-dependent DNA repair pathways

Article https://doi.org/10.1038/s41467-025-67216-3 Molecular basis of XPF-ERCC1 targeting to SLX4-dependent DNA repair pathways Received: 26 February 2025 Accepted: 24 November 2025 1234567890():,; 1234567890():,; Check for updates Junjie Feng 1, Peter R. Martin 2, Szymon Kowalski2,3, Maxime Lecot Nora B. Cronin 4, Teige Matthews-Palmer 1, Wojciech Niedzwiedz Basil J. Greber 1 2,5 , 2 & The preservation and faithful propagation of genetic information is essential for all life forms and depends on cellular pathways that enable replication, recombination, and repair of DNA. The multifunctional XPF-ERCC1 DNA endonuclease complex acts in several DNA repair pathways and interacts with numerous partner proteins and large DNA repair assemblies, including the nucleotide excision repair machinery and the SMX tri-endonuclease complex. Here, we report structures of XPF-ERCC1 in complex with the DNA repair factors SLX4 and SLX4IP, thereby identifying key residues responsible for direct interactions with XPF-ERCC1. When introduced into human cells, point mutations in these interfaces impair the interactions between XPF-ERCC1 and SLX4 or SLX4IP, and disruption of the XPF-SLX4IP interface leads to cis-platin sensitivity. Furthermore, our data reveal the structure of the human XPFERCC1-SLX4IP-SLX4330-555 complex with DNA bound at its active site, and they complete the structural characterisation of molecular interactions required to assemble the SMX complex. Maintenance of genome integrity is critical for all cellular life. Therefore, cells have evolved pathways that remove or mitigate DNA damage and facilitate interconversion of unstable DNA structures that arise during cellular processes such as DNA replication or recombination. Nucleolytic enzymes called structure-specific or structureselective endonucleases are instrumental in several DNA repair pathways1. The sites of action of structure-specific endonucleases are determined by structural features of the DNA, most typically junctions between single-stranded and double-stranded DNA (ssDNA and dsDNA, respectively), rather than by DNA target sequences1. Due to this property, the activity of structure-specific endonucleases needs to be tightly controlled in time and space to avoid unwanted DNA incisions that can have disastrous consequences for the cell2, such as chromosome pulverisation caused by premature activation of the SLX1-SLX4-MUS81-EME1 endonuclease complex3. Conversely, failure to properly target and activate structure-specific endonucleases can impair important cellular processes, including DNA repair, DNA replication, and DNA segregation. XPF-ERCC1 (xeroderma pigmentosum complementation group F and excision repair cross-complementing group 1) is a structure-specific endonuclease complex that specifically cleaves 3’-flaps, i.e. junctions between a DNA duplex and a singlestranded 3’-overhang, and damage-containing DNA bubbles on the 5’side of the lesion4,5. Mutations affecting the complex are causative of human disease, including Fanconi anaemia6,7 and the cancer-prone UVsensitive syndrome xeroderma pigmentosum from which the name of the catalytic XPF subunit is derived4,8,9. Concurrently, inhibition of XPFERCC1 has been suggested as a possible mechanism for sensitising cancer cells to cis-platin treatment10. The enzymatic activity of XPF-ERCC1 is employed in several DNA repair and genome maintenance processes, including nucleotide 1 Division of Structural Biology, The Institute of Cancer Research, Chester Beatty Laboratories, London, UK. 2Division of Cell and Molecular Biology, The Institute of Cancer Research, Chester Beatty Laboratories, London, UK. 3Department of Pharmacology, Faculty of Medicine, Medical University of Gdańsk, Gdańsk, Poland. 4London Consortium for High Resolution Cryo-EM, The Francis Crick Institute, London, UK. 5Present address: Faculty of Medicine of Rennes, University of Rennes, F-35043 Rennes, France and Molecular Oncology, Institut Curie, PSL Research University, CNRS, UMR144, Paris, France. e-mail: Nature Communications | (2026)17:522 1 Article https://doi.org/10.1038/s41467-025-67216-3 excision repair (NER)2, inter-strand crosslink (ICL) repair6,7,11, homologous recombination12, alternative lengthening of telomeres (ALT)13, and TRF2-mediated telomere shortening14–16. The recruitment of XPFERCC1 to its DNA target sites in these pathways depends on interactions with other DNA repair factors that provide, albeit sometimes indirectly, a target-recognition function and act to enhance XPF-ERCC1 endonuclease activity. Among these XPF-ERCC1 recruiting DNA repair factors is SLX417, a 1834 aa-long scaffold protein that additionally binds the endonuclease SLX1 and the endonuclease complex MUS81EME118–23. This complex is called SMX (SLX-MUS-XPF) in its assembled form18. The activities of the participating endonucleases are enhanced by complex formation18,21,24,25, and subsets of the three nucleases are coordinated to enable their functions in several DNA repair pathways. Specifically, XPF-ERCC1 acts in an SLX4-dependent manner to unhook inter-strand cross-links in ICL repair26–29 and to release stalled replication forks for homologous recombination30. SLX1-SLX4 and MUS81-EME1 form a Holliday junction resolvase18–22 that is further activated by a non-enzymatic contribution from XPFERCC118, and MUS81-EME1 within SMX cleaves replication forks18, contributing to maintenance of common fragile sites. The molecular details of the SLX4-SLX1 and SLX4-MUS81-EME1 interactions have been elucidated by X-ray crystallographic and nuclear magnetic resonance (NMR) structures (refs. 31–34 and the unpublished X-ray crystal structure PDB ID 7BU5). In contrast, structural insight into the XPFERCC1 recruiting activity of SLX4 has remained elusive. Biochemical a 205 220 1 XPF RecA1 631 680 341 Helical 819 845 906 916 1 100 ERCC1 Nuclease HhH2 RecA2 293 361 409 555 2x MLR UBZ 1 SLX4 and cell biological characterisation revealed that SLX4 residues 529550 (refs. 27,35,36) residing within an SLX4 domain near the MUS312/ MEI9 interaction-like region (MLR)11,20,22 are required for the interaction (Fig. 1a). In addition to formation of the SMX complex, SLX4 and its partner endonucleases interact with numerous additional DNA repair factors that enable the complex or its components to act in additional DNA repair and genome maintenance pathways. One of these is SLX4IP (C20orf94)22, a protein involved in the ALT and ICL repair pathways27,37. In ALT, a telomerase-independent strategy used by certain cancer cells to achieve replicative immortality, SLX4IP appears to balance the activity of the Bloom syndrome helicase (BLM) and SMX by repressing BLM37. Within SMX-related complexes, SLX4IP has been suggested to interact with SLX4, with XPF, or with both XPF and SLX422,27,37. However, the molecular basis of these interactions and the mechanism of action of the complex in ALT and ICL repair have not been elucidate (...truncated)