Figure 1. HMCES‐DNA‐protein crosslinks are reversible.

1. SRAP domain sequence alignment highlighting key active site residues in H. sapiens, X. laevis, S. cerevisiae and E. coli HMCES homologues (Cys2 = orange, Glu127 = red and His210 = green).
2. Proposed reaction mechanism of SRAP domain crosslinking to an AP site.
3. Crystal structure of HMCES' active site crosslinked to an AP site. PDB: 6OE7 (Halabelian et al, 2019). DNA is shown in grey. Active site residues are coloured as in (A). Interatomic distances (Å) are labelled.
4. Schematic of the assay shown in (E) and (G). HMCES^FL and HMCES^SRAP (WT or active site variants) were incubated for 1 h at 37°C with a Cy5‐labelled 30mer oligonucleotide containing either a dT or an AP site at position 15. Afterwards, non‐crosslinked HMCES was inactivated by heat denaturation at 60°C for 5 min. A second 6‐FAM‐labelled 30mer oligonucleotide containing an AP site was added and formation of 6‐FAM DPCs was assessed after an additional incubation for 120 min.
5. HMCES^FL‐ and HMCES^SRAP‐WT and HMCES^SRAP‐C2S‐DPC formation with Cy5‐ and 6‐FAM‐oligonucleotides was analysed using denaturing SDS–PAGE. Incised DNA is caused by spontaneous hydrolysis of the AP site.
6. Quantification of DPC formation assays shown in (E), left panel: DPC formation to Cy5 oligonucleotide, right panel: DPC formation to 6‐FAM oligonucleotide.
7. DPC formation of HMCES^SRAP‐WT and variants (E127A or H210A) with Cy5‐ and 6‐FAM‐oligonucleotides was analysed using denaturing SDS–PAGE. Incised DNA is caused by spontaneous hydrolysis of the AP site.
8. Quantification of DPC formation assays shown in (G), left panel: DPC formation to Cy5 oligonucleotide, right panel: DPC formation to 6‐FAM oligonucleotide.

Data information: Bar graphs in (F) and (H) show the mean of three independent experiments ± SD. Two WT data points are common between (F) and (H).

Source data are available online for this figure.