Journal: Nature Communications

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

doi: 10.1038/s41467-025-67216-3

Figure Lengend Snippet: a Schematic representation of the domain organisation of XPF, ERCC1, SLX4IP, and SLX4 (domain boundaries according to , or structural results from this work). SLX4 variants expressed for structural and biochemical experiments are indicated. Abbreviations: HhH helix-hairpin-helix, NLD nuclease-like domain, UBZ ubiquitin-binding zinc finger type 4, MLR MUS312-MEI9 interaction-like region, BTB broad-complex, tramtrack and bric-a-brac, SAP SAF-AB, acinus and PIAS, CCD conserved C-terminal domain. b Cryo-EM reconstruction of the XPF-ERCC1-SLX4IP-SLX4 330-555 complex. c Structure of SLX4 residues 526-552 bound to XPF. d Structure of SLX4IP bound to XPF.

Article Snippet: Primary antibodies used were: GFP (Roche Cat# 11814460001, RRID:AB_390913, 1:500), MUS81 (Santa Cruz Biotechnology Cat# sc-47692, RRID:AB_2147129,1:500), ERCC1 (Santa Cruz Biotechnology Cat# sc-17809, RRID:AB_2278023,1:500), SLX4 (MRC-PPU Cat# S714C, RRID:AB_2752254, 1:500), SLX1 (Proteintech Cat# 21158-1-AP, RRID:AB_2752255, 1:500), EME1 (Santa Cruz Biotechnology Cat# sc-393363, 1:500), XPF (Bethyl Cat# A301-315A, RRID:AB_938089 1:500), and SLX4IP (Santa Cruz Biotechnology Cat# sc-377066, RRID:AB_2752253).

Techniques: Ubiquitin Proteomics, Binding Assay, Cryo-EM Sample Prep

Journal: Nature Communications

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

doi: 10.1038/s41467-025-67216-3

Figure Lengend Snippet: a Y-fork DNA substrate used to assay activity of XPF-ERCC1 complexes. The preferred cleavage site of XPF-ERCC1 is denoted by an arrow and liberates a 23-nt DNA fragment with a 3’-Cy3 fluorophore used for detection of the product. b Comparison of nuclease activity of XPF-ERCC1, XPF-ERCC1-SLX4IP, XPF-ERCC1-SLX4IP-SLX4 330-555 , and XPF-ERCC1-SLX4 330-555 . Conversion of uncleaved input substrate (a) into product (23-nt fragment) was monitored by detection of Cy3 fluorescence. SLX4IP has no impact on activity, while the presence of SLX4 in the complex increases cleavage, as evidenced by the more rapid disappearance of the band corresponding to the intact substrate. To facilitate visualisation, the 5 min time points are marked with red dots. c Coomassie stained SDS-PAGE gel of protein sample processing control (before final 10x dilution into the nuclease reaction) to confirm that equivalent amounts of endonuclease were present in all samples. Two additional repeats of the experiment shown in panels b and c are provided in Supplementary Fig. . d Y-fork DNA substrate used to assemble an XPF-ERCC1-SLX4IP-SLX4 330-555 -DNA complex for cryo-EM. Bonds protected against endonucleolytic cleavage by phosphorothioate linkages are indicated by asterisks (*). Source Data are provided as a file.

Article Snippet: Primary antibodies used were: GFP (Roche Cat# 11814460001, RRID:AB_390913, 1:500), MUS81 (Santa Cruz Biotechnology Cat# sc-47692, RRID:AB_2147129,1:500), ERCC1 (Santa Cruz Biotechnology Cat# sc-17809, RRID:AB_2278023,1:500), SLX4 (MRC-PPU Cat# S714C, RRID:AB_2752254, 1:500), SLX1 (Proteintech Cat# 21158-1-AP, RRID:AB_2752255, 1:500), EME1 (Santa Cruz Biotechnology Cat# sc-393363, 1:500), XPF (Bethyl Cat# A301-315A, RRID:AB_938089 1:500), and SLX4IP (Santa Cruz Biotechnology Cat# sc-377066, RRID:AB_2752253).

Techniques: Activity Assay, Comparison, Fluorescence, Staining, SDS Page, Control, Cryo-EM Sample Prep

Journal: Nature Communications

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

doi: 10.1038/s41467-025-67216-3

Figure Lengend Snippet: a Cryo-EM map of the XPF-ERCC1-SLX4IP-SLX4 330-555 -DNA complex. XPF is shown in cyan, ERCC1 in blue, SLX4 in orange, SLX4IP in yellow, the scissile DNA strand in light green, and the non-scissile strand in dark green. b Atomic model of the XPF-ERCC1-SLX4IP-SLX4 330-555 -DNA complex (colours as in b ). c Surface view of the proteins in the complex with residues in proximity to DNA (<3.5 Å) shown in pink. DNA is not shown. d Close-up view of the DNA contacts with the ERCC1 HhH 2 domain. e Close-up view of the DNA contacts with the XPF nuclease and RecA1 domains. Manganese ions bound in the active site shown as spheres. f View of the XPF active site with two metal ions coordinated by negatively charged residues (shown in cyan) and a DNA backbone phosphate.

Article Snippet: Primary antibodies used were: GFP (Roche Cat# 11814460001, RRID:AB_390913, 1:500), MUS81 (Santa Cruz Biotechnology Cat# sc-47692, RRID:AB_2147129,1:500), ERCC1 (Santa Cruz Biotechnology Cat# sc-17809, RRID:AB_2278023,1:500), SLX4 (MRC-PPU Cat# S714C, RRID:AB_2752254, 1:500), SLX1 (Proteintech Cat# 21158-1-AP, RRID:AB_2752255, 1:500), EME1 (Santa Cruz Biotechnology Cat# sc-393363, 1:500), XPF (Bethyl Cat# A301-315A, RRID:AB_938089 1:500), and SLX4IP (Santa Cruz Biotechnology Cat# sc-377066, RRID:AB_2752253).

Techniques: Cryo-EM Sample Prep

Journal: Nature Communications

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

doi: 10.1038/s41467-025-67216-3

Figure Lengend Snippet: a Left: Cryo-EM map of the XPF-ERCC1-SLX4IP-SLX4 330-555 -DNA complex. Three SLX4 segments (orange) are visualised and labelled. Right: Cryo-EM map of the DNA-free complex from the same data collection shown in the same orientation. Only SLX4 residues 526-552 are visualised, in line with the higher-resolution reconstructions shown in Fig. . Maps are shown without any b-factor sharpening applied to facilitate visualisation of weaker densities. b Schematic depiction of the sequence elements and domains contained in the SLX4 constructs used for in vitro endonuclease assays of XPF-ERCC1-SLX4IP-SLX4 complex variants. c In-vitro endonuclease assay comparing the activity of XPF-ERCC1 to the activities of different variants of the XPF-ERCC1-SLX4IP-SLX4 330-555 complex. Conversion of the input substrate (see Fig. ) into 23-nt product was monitored by detection of Cy3 fluorescence. To facilitate visualisation, 10 min time points are marked with a red dot. The removal of the UBZ-2 domain and the addition of the BTB domain do not have an appreciable effect on endonuclease activity. Removal of the sequence elements that are solely visualised in the DNA-bound XPF-ERCC1-SLX4IP-SLX4 330-555 complex strongly reduces cleavage activity. Two additional experimental repeats and all protein sample processing controls are provided in Supplementary Fig. . Source Data are provided as a file.

Article Snippet: Primary antibodies used were: GFP (Roche Cat# 11814460001, RRID:AB_390913, 1:500), MUS81 (Santa Cruz Biotechnology Cat# sc-47692, RRID:AB_2147129,1:500), ERCC1 (Santa Cruz Biotechnology Cat# sc-17809, RRID:AB_2278023,1:500), SLX4 (MRC-PPU Cat# S714C, RRID:AB_2752254, 1:500), SLX1 (Proteintech Cat# 21158-1-AP, RRID:AB_2752255, 1:500), EME1 (Santa Cruz Biotechnology Cat# sc-393363, 1:500), XPF (Bethyl Cat# A301-315A, RRID:AB_938089 1:500), and SLX4IP (Santa Cruz Biotechnology Cat# sc-377066, RRID:AB_2752253).

Techniques: Cryo-EM Sample Prep, Sequencing, Construct, In Vitro, Activity Assay, Fluorescence

Journal: Nature Communications

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

doi: 10.1038/s41467-025-67216-3

Figure Lengend Snippet: a SLX4IP and XPF residues mediating the SLX4IP-XPF interaction are coloured green (left) and red (right), respectively. b Mapping of mutations that have been reported to disrupt the SLX4IP-XPF interaction. c Conformation of XPF in the absence of SLX4IP. d Conformation of XPF after strand exchange involving XPF residues 542–556 upon SLX4IP binding. e Interactions mediated by XPF residues 542–549. f Conformational change in XPF residues 557–573 upon binding of SLX4IP (light green: free XPF; dark green: SLX4IP-bound XPF).

Article Snippet: Primary antibodies used were: GFP (Roche Cat# 11814460001, RRID:AB_390913, 1:500), MUS81 (Santa Cruz Biotechnology Cat# sc-47692, RRID:AB_2147129,1:500), ERCC1 (Santa Cruz Biotechnology Cat# sc-17809, RRID:AB_2278023,1:500), SLX4 (MRC-PPU Cat# S714C, RRID:AB_2752254, 1:500), SLX1 (Proteintech Cat# 21158-1-AP, RRID:AB_2752255, 1:500), EME1 (Santa Cruz Biotechnology Cat# sc-393363, 1:500), XPF (Bethyl Cat# A301-315A, RRID:AB_938089 1:500), and SLX4IP (Santa Cruz Biotechnology Cat# sc-377066, RRID:AB_2752253).

Techniques: Binding Assay