Fig 2: SPRTN is a DNA binding protease.(A) Western blot analysis of GFP-SPRTN expression. GFP-SPRTN and corresponding mutants were immunoprecipitated after 16 hr of transient transfection in 293T-HEK cells. Arrows indicate full-length (FL) or cleaved fragments. (B) Western blot analysis of histone H3 cleavage in cells overexpressing GFP-SPRTN, GFP-SPRTN-E112A or GFP-SPRTN-∆C. (C) EMSA of full-length recombinant SPRTN or SPRTN-∆C (a.a. 1–216) and 0.25 uM 6-FAM-39-mer single stranded oligodeoxynucleotide (6-FAM-ssODN) or (D) 0.25 uM 6’FAM-39-mer double stranded oligonucleotide (6-FAM-dsODN) .DOI: http://dx.doi.org/10.7554/eLife.21491.006

Fig 3: SPRTN mutant proteins from Ruijs-Aalfs syndrome patients are hypomorphic for DPC removal.(A) Comparison of SPRTN-Y117C and SPRTN-∆C mutant proteins found in patients with Ruijs-Aalfs syndrome. The NLS sequence highlights the amino acid conservation in vertebrate SPRTN species. (B) Fluorescent expression of GFP-SPRTN and mutant proteins in live U2OS cells. (C) ICE assay of DNA isolated from SPRTN control, SPRTN-KO cells or SPRTN-KO cells reconstituted with SPRTN-WT, SPRTN-∆C+NLS, or SPRTN-R408A L411A. Cells were either untreated or treated for 30 min with 25 uM Etoposide and immediately subjected to ICE assay or 1 and 2 hr after washing out Etoposide (recovery), as in Figure 2C. Western blot indicates SPRTN levels after reconstitution of SPRTN-KO MEFS. (D) Representative flow cytometry analysis of control LCL cells and patient (B-II-1) LCL cells treated with formaldehyde, Etoposide or CPT for 0, 1 or 4 hr. Cells were collected at indicated time points and processed for γ-H2AX staining. Untreated controls are indicated in black for comparison to treated cells. 10,000 cells were scored for each experiment. (E) ICE assay of DNA isolated from control or B-II-1 cells that were either untreated or treated with 25 uM Etoposide for the indicated times as in Figure 2C and 5C.DOI: http://dx.doi.org/10.7554/eLife.21491.011

Fig 4: SPRTN fails to complement Wss1 activity in yeast.(A) Comparison of SPRTN and Wss1 domain organization. (B) Comparison of SprT-like domains from selected SPRTN-containing organisms. Amino acid highlighted in red corresponds to the tyrosine mutated in patients with Ruijs-Aalfs syndrome (SPRTN-Y117C), identical amino acids are in blue and similar amino acids are in yellow. The catalytic glutamic acid and zinc-coordinating histidines are indicated by bold text. (C) Yeast spot assay. Five-fold dilutions of wildtype, tdp1∆, and wss1∆ tdp1∆ cells. wss1∆ tdp1∆ cells harboring empty plasmid (--- = pRS415) or plasmid encoding wild-type SPRTN or corresponding mutants under the endogenous Wss1 promoter. Cells were spotted on CSM-LEU plates with 2 ug/mL doxycycline or 2 ug/mL doxycycline and 40 uM CPT. Doxycycline is used to acutely deplete Wss1 protein levels in tdp1∆ cells to effectively obtain wss1∆ tdp1∆ cells.DOI: http://dx.doi.org/10.7554/eLife.21491.002

Fig 5: Model of SPRTN removal of DNA-protein crosslinks.DNA-protein crosslinks may arise stochastically as a consequence of normal metabolic processes occurring at or in the vicinity of DNA or by exogenous sources such as Etoposide. DPCs are resolved by SPRTN to allow for unperturbed and timely DNA replication. When SPRTN is absent or when its activity is compromised, such as in patients with Ruijs-Aalfs syndrome (SPRTN-Y117C or SPRTN-∆C), DPCs can persist and perturb DNA replication and transcription (not shown). DPCs, if not resolved, can lead to genomic instability, tumorigenesis and contribute to accelerated aging.DOI: http://dx.doi.org/10.7554/eLife.21491.013