Fig. 3: XPF-ERCC1 endonuclease activity in the presence of SLX4IP and SLX4330-555.
From: Molecular basis of XPF-ERCC1 targeting to SLX4-dependent DNA repair pathways
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-SLX4330-555, and XPF-ERCC1-SLX4330-555. 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. 5. d Y-fork DNA substrate used to assemble an XPF-ERCC1-SLX4IP-SLX4330-555-DNA complex for cryo-EM. Bonds protected against endonucleolytic cleavage by phosphorothioate linkages are indicated by asterisks (*). Source Data are provided as a Source data file.
