Extended Data Fig. 12: Related to Fig. 4. In vitro cruciform unwinding and rescue of 53BP1 foci.
From: Comprehensive interrogation of synthetic lethality in the DNA damage response
a, Polyacrylamide gels showing recombinant SMARCAL1, SMARCAL1 ΔRBM and FANCM (n = 1). b, Schematic of the different substrates used in the unwinding assays. c, Gels validating secondary structure formation. Incubation with the structure-specific T7 Endonuclease I and SspI results in the production of bands at the expected positions of 606 and 2,120 bp (n = 1). The SspI restriction site is 606 bp away from the cruciform site. Incubation with EcoRI produces a linear DNA fragment with PUC19 and PUC19_chr3. On PUC19_TA, the EcoRI sites are at the apex of the cruciform, and hence refractory to cleavage when a cruciform is present. d, Representative cruciform unfolding assay on the PUC19_chr3 substrate showing SMARCAL1, SMARCAL1 ΔRBM and FANCM prevent DNA cutting by T7 endonuclease I (n = 2). e, Representative unwinding assay of PUC19_chr3 in the presence and absence of RPA (20 nM). SMARCAL1 ΔRBM shows a reduced activity in the presence of RPA at lower concentrations. Quantified for 30 nM in f. g, Quantification of 53BP1 foci in the indicated cells. At least 100 cells were quantified per condition. Red line denotes the mean. h, RT-qPCR data of cells transduced with sgCTRL, sgERCC1, sgERCC4, sgMUS81, sgGEN1 show knockdown of mRNA transcripts for each sgRNA. Error bars represent mean ± s.d. of three experimental replicates.
