Fig. 5: Recombinant ERCC6L2 binds and melts DNA substrates with short overhangs.
From: ERCC6L2 ensures repair fidelity for staggered-end DNA double-strand breaks
a Schematic of the different DNA substrates for binding and melting assays. dsDNA, double-stranded DNA; ssDNA, single-stranded DNA; bp, base pair; nt, nucleotide. b Electrophoretic mobility shift assay (EMSA) with increasing concentrations of ERCC6L2 with the substrates depicted in (a). Protein-bound DNA exhibits an upwards gel shift. c Quantification of protein-bound DNA from (b) (n = 3 biological replicates). d DNA melting assay with increasing concentrations of ERCC6L2 in the presence of RPA to prevent reannealing of DNA. Asterisk denotes a substrate truncation species in the Y-structure DNA condition. e. Quantification of single-stranded DNA (ssDNA) product from (d) (n = 5 biological replicates). f DNA melting assays of the 3’ overhang substrate to assess activity of ERCC6L2 helicase function in the presence of ATP, non-hydrolyzable AMP-PNP, no ATP, or EDTA. g Quantification of ssDNA product from (f) (n = 3–10 biological replicates). h DNA melting assay using either wild-type (WT) or K154A ERCC6L2 with the 3’ overhang substrate assessing activity in the presence of ATP or non-hydrolyzable AMP-PNP. Assay performed with 60 mM NaCl instead of 20 mM NaCl (n = 3–4 biological replicates). i Quantification of ssDNA product at 5 nM ERCC6L2 from experiments such as in (h). j Model for the role of ERCC6L2 in staggered DSB repair. Bar graphs in (c, e, g, i) correspond to mean ± SD. Statistical significance was determined with a two-tailed Welch’s t-test in (e, g, i). Source data are provided as a Source Data file.
