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. 2023 Jul 31;42(18):e113360. doi: 10.15252/embj.2022113360

Figure EV2. Release of HMCES‐DPCs is determined by DNA context. Related to Fig 3 .

Figure EV2

  1. Fluorescence polarization measurements of Cy5‐labelled ssDNA (25 nM) incubated with increasing concentrations of HMCESSRAP‐WT, HMCESSRAP‐R98E, HMCESSRAP‐E127A or HMCESSRAP‐R98E/E127A for 20 min on ice prior to measuring fluorescence polarization.
  2. Non‐covalent DNA binding of indicated HMCESSRAP variants was assessed by electrophoretic mobility shift assay. A Cy5‐labelled 30‐mer ssDNA (0.1 μM) was incubated with HMCESSRAP (0, 0.125, 0.5 or 2 μM) for 20 min at 4°C prior to analysis by native PAGE.
  3. DPC reversal kinetics of indicated HMCESSRAP variants in dsDNA. A corresponding reverse oligonucleotide was annealed to HMCESSRAP‐DPCs, before incubation for the indicated amount of time at 37°C prior to separation by denaturing SDS–PAGE.
  4. Quantification of DPC reversal assays shown in (C).

Data information: Data in (A) and (D) represent the mean of three independent experiments ± SD.