The cytotoxicity of DNA-protein crosslinks (DPCs) is largely ascribed to their ability to block the progression of DNA replication. DPCs frequently occur in cells, either as a consequence of metabolism or exogenous agents, but the mechanism of DPC repair is not completely understood. Here, we characterize SPRTN as a specialized DNA-dependent and DNA replication-coupled metalloprotease for DPC repair. SPRTN cleaves various DNA binding substrates during S-phase progression and thus protects proliferative cells from DPC toxicity. Ruijs-Aalfs syndrome (RJALS) patient cells with monogenic and biallelic mutations in SPRTN are hypersensitive to DPC-inducing agents due to a defect in DNA replication fork progression and the inability to eliminate DPCs. We propose that SPRTN protease represents a specialized DNA replication-coupled DPC repair pathway essential for DNA replication progression and genome stability. Defective SPRTN-dependent clearance of DPCs is the molecular mechanism underlying RJALS, and DPCs are contributing to accelerated aging and cancer.

Correct replication of the genome and protection of its integrity are essential for cell survival. In a high-throughput screen studying H2AX phosphorylation, we identified Wee1 as a regulator of genomic stability. Wee1 down-regulation not only induced H2AX phosphorylation but also triggered a general deoxyribonucleic acid (DNA) damage response (DDR) and caused a block in DNA replication, resulting in accumulation of cells in S phase. Wee1-deficient cells showed a decrease in replication fork speed, demonstrating the involvement of Wee1 in DNA replication. Inhibiting Wee1 in cells treated with short treatment of hydroxyurea enhanced the DDR, which suggests that Wee1 specifically protects the stability of stalled replication forks. Notably, the DDR induced by depletion of Wee1 critically depends on the Mus81-Eme1 endonuclease, and we found that codepletion of Mus81 and Wee1 abrogated the S phase delay. Importantly, Wee1 and Mus81 interact in vivo, suggesting direct regulation. Altogether, these results demonstrate a novel role of Wee1 in controlling Mus81 and DNA replication in human cells.

The forkhead transcription factor FOXM1 has a key role in DNA damage response, and its deregulated overexpression is associated with genotoxic drug resistance in breast cancer. However, little is known about the posttranslational mechanisms by which FOXM1 expression is regulated by genotoxic agents and how they are deregulated in resistant cells. Initial co-immunoprecipitation studies verified previous proteomic analysis finding that the OTUB1 is a novel FOXM1-interacting protein. Western blot analysis showed that both OTUB1 and FOXM1 expression reduced upon genotoxic agent treatment in MCF-7 cells, but remained relatively constant in resistant cells. FOXM1 expression reduced upon OTUB1 depletion by siRNA and increased with OTUB1 overexpression in MCF-7 cells, arguing that OTUB1 positively regulates FOXM1 expression. In agreement, co-immunoprecipitation experiments demonstrated that FOXM1 expression is associated with OTUB1 binding but inversely correlates with conjugation to the protein degradation-associated Lys-48-linked ubiquitin-chains. Overexpression of wild-type (WT) OTUB1, but not the OTUB1(C91S) mutant, disrupted the formation of Lys48-linked ubiquitin-conjugates on FOXM1. Importantly, knockdown of OTUB1 by siRNA resulted in an increase in turnover of FOXM1 in MCF-7 cells treated with the protein synthesis inhibitor cycloheximide, whereas overexpression of WT OTUB1, but not the OTUB1(C91S) mutant, significantly enhances the half-life of FOXM1. In addition, proliferative and clonogenic assays also show that OTUB1 can enhance the proliferative rate and epirubicin resistance through targeting FOXM1, as OTUB1 has little effect on FOXM1-deficient cells. The physiological relevance of the regulation of FOXM1 by OTUB1 is further underscored by the significant correlations between FOXM1 and OTUB1 expression in breast cancer patient samples. Cox-regression survival analysis indicates that OTUB1 overexpression is linked to poorer outcome in particular in patients treated with chemotherapy. Collectively, these data suggest that OTUB1 limits the ubiquitination and degradation of FOXM1 in breast cancer and has a key role in genotoxic agent resistance.