Fig. 1: Ubiquitylation of DPCs promotes their cleavage by SPRTN.
From: Allosteric activation of the SPRTN protease by ubiquitin maintains genome stability
a Schematic of SPRTN’s domain structure and truncated variants, featuring motif interacting with ubiquitin (MIU), protease domain, zinc-binding domain (ZBD), basic region (BR), SHP box for p97-binding, PCNA-interacting motif (PIP) and ubiquitin-binding zinc finger (UBZ). SPRTNΔC is caused by a frameshift mutation resulting in a variant composed of SPRTN’s N-terminal 240 residues followed by eight additional amino acids (X8). b Schematic of HMCESSRAP ubiquitylation to generate DPCs shown in e, f, Fig. 4 and Supplementary Fig. 5b and 6b. HMCESSRAP-Ub(G76V)-3C-FKBP was incubated with FRB-E3 + E2 (K48 or K63) in the presence of ubiquitin, rapamycin, ubiquitin-E1 and ATP for 2 h (K63) or 6.5 h (K48) at 30 °C. After cleavage of the FKBP-tag via 3C-protease, ubiquitylated HMCESSRAP was purified by reverse immobilized metal affinity chromatography (IMAC) and size-exclusion chromatography (SEC). c Mass spectrometry analysis of ubiquitin linkages formed by ubiquitylation of HMCESSRAP as shown in (b). Bar chart shows the mean ± SD of three biological replicates. d Schematic of the generation of HMCESSRAP-DPCs. HMCESSRAP was incubated for 30 min at 37 °C with a Cy5-labeled 30nt oligonucleotide containing a dU at position 15 and UDG. After crosslinking a complementary 15nt reverse oligonucleotide was annealed to form a ssDNA-dsDNA junction. e Indicated HMCESSRAP-DPCs (10 nM) were incubated alone or in the presence of FANCJ (100 nM) and indicated concentrations of SPRTN (1-100 nM) for 1 h at 30 °C. Quantification: bar graphs represent the mean ± SD of three independent experiments. All samples derive from the same experiment and gels were processed in parallel. Values for cleavage of unmodified HMCESSRAP-DPC are the same as in Supplementary Fig. 1b. Source data are provided as a Source Data file. f Indicated HMCESSRAP-DPCs (10 nM) were incubated alone or in the presence of FANCJ (100 nM) and indicated concentrations of SPRTN or SprT-BR (1-100 nM) for 1 h at 30 °C. Quantification: bar graphs represent the mean ± SD of three independent experiments. All samples derive from the same experiment and gels were processed in parallel. Source data are provided as a Source Data file.
