DNA Structure-Specific Cleavage of DNA-Protein Crosslinks by the SPRTN Protease.

Repair of covalent DNA-protein crosslinks (DPCs) by DNA-dependent proteases has emerged as an essential genome maintenance mechanism required for cellular viability and tumor suppression. However, how proteolysis is restricted to the crosslinked protein while leaving surrounding chromatin proteins unharmed has remained unknown. Using defined DPC model substrates, we show that the DPC protease SPRTN displays strict DNA structure-specific activity. Strikingly, SPRTN cleaves DPCs at or in direct proximity to disruptions within double-stranded DNA. In contrast, proteins crosslinked to intact double- or single-stranded DNA are not cleaved by SPRTN. NMR spectroscopy data suggest that specificity is not merely affinity-driven but achieved through a flexible bipartite strategy based on two DNA binding interfaces recognizing distinct structural features. This couples DNA context to activation of the enzyme, tightly confining SPRTN's action to biologically relevant scenarios.

The Aspartic Protease Ddi1 Contributes to DNA-Protein Crosslink Repair in Yeast.

Naturally occurring or drug-induced DNA-protein crosslinks (DPCs) interfere with key DNA transactions if not repaired in a timely manner. The unique family of DPC-specific proteases Wss1/SPRTN targets DPC protein moieties for degradation, including stabilized topoisomerase-1 cleavage complexes (Top1ccs). Here, we describe that the efficient DPC disassembly requires Ddi1, another conserved predicted protease in Saccharomyces cerevisiae. We found Ddi1 in a genetic screen of the tdp1 wss1 mutant defective in Top1cc processing. Ddi1 is recruited to a persistent Top1cc-like DPC lesion in an S phase-dependent manner to assist in the eviction of crosslinked protein from DNA. Loss of Ddi1 or its putative protease activity hypersensitizes cells to DPC trapping agents independently from Wss1 and 26S proteasome, implying its broader role in DPC repair. Among the potential Ddi1 targets, we found the core component of Pol II and show that its genotoxin-induced degradation is impaired in ddi1. We propose that the Ddi1 protease contributes to DPC proteolysis.

RAD51-WSS1-dependent genetic pathways are essential for DNA-protein crosslink repair and pathogenesis in Candida albicans.

Genetic analyses in Saccharomyces cerevisiae suggest that nucleotide excision repair (NER), homologous recombination (HR), and protease-dependent repair pathways coordinately function to remove DNA-protein crosslinks (DPCs) from the genome. DPCs are genomic cytotoxic lesions generated because of the covalent linkage of proteins with DNA. Although NER and HR processes have been studied in pathogenic Candida albicans, their roles in DPC repair (DPCR) are yet to be explored. Proteases like Wss1 and Tdp1 (tyrosyl-DNA phosphodiesterase-1) are known to be involved in DPCR; however, Tdp1 that selectively removes topoisomerase-DNA complexes is intrinsically absent in C. albicans. Therefore, the mechanism of DPCR might have evolved differently in C. albicans. Herein, we investigated the interplay of three genetic pathways and found that RAD51-WSS1-dependent HR and protease-dependent repair pathways are essential for DPC removal, and their absence caused an increased rate of loss of heterozygosity in C. albicans. RAD1 but not RAD2 of NER is critical for DPCR. In addition, we observed truncation of chromosome #6 in the cells defective in both RAD51 and WSS1 genes. While the protease and DNA-binding activities are essential, a direct interaction of Wss1 with the eukaryotic DNA clamp proliferating cell nuclear antigen is not a requisite for the function of Wss1. DPCR-defective C. albicans cells exhibited filamentous morphology, reduced immune cell evasion, and attenuation in virulence. Thus, we concluded that RAD51-WSS1-dependent DPCR pathways are essential for genome stability and candidiasis development. Since no vaccine against candidiasis is available for human use yet, we propose to explore DPCR-defective attenuated strains (rad51ΔΔwss1ΔΔ and rad2ΔΔrad51ΔΔwss1ΔΔ) for whole-cell vaccine development.

Mechanism and Regulation of DNA-Protein Crosslink Repair by the DNA-Dependent Metalloprotease SPRTN.

Covalent DNA-protein crosslinks (DPCs) are toxic DNA lesions that interfere with essential chromatin transactions, such as replication and transcription. Little was known about DPC-specific repair mechanisms until the recent identification of a DPC-processing protease in yeast. The existence of a DPC protease in higher eukaryotes is inferred from data in Xenopus laevis egg extracts, but its identity remains elusive. Here we identify the metalloprotease SPRTN as the DPC protease acting in metazoans. Loss of SPRTN results in failure to repair DPCs and hypersensitivity to DPC-inducing agents. SPRTN accomplishes DPC processing through a unique DNA-induced protease activity, which is controlled by several sophisticated regulatory mechanisms. Cellular, biochemical, and structural studies define a DNA switch triggering its protease activity, a ubiquitin switch controlling SPRTN chromatin accessibility, and regulatory autocatalytic cleavage. Our data also provide a molecular explanation on how SPRTN deficiency causes the premature aging and cancer predisposition disorder Ruijs-Aalfs syndrome.

Ubiquitin-directed AAA+ ATPase p97/VCP unfolds stable proteins crosslinked to DNA for proteolysis by SPRTN.

The protease SPRTN degrades DNA-protein crosslinks (DPCs) that threaten genome stability. SPRTN has been connected to the ubiquitin-directed protein unfoldase p97 (also called VCP or Cdc48), but a functional cooperation has not been demonstrated directly. Here, we biochemically reconstituted p97-assisted proteolysis with purified proteins and showed that p97 targets ubiquitin-modified DPCs and unfolds them to prepare them for proteolysis by SPRTN. We demonstrate that purified SPRTN alone was unable to degrade a tightly-folded Eos fluorescent reporter protein even when Eos was crosslinked to DNA (Eos-DPC). However, when present, p97 unfolded poly-ubiquitinated Eos-DPC in a manner requiring its ubiquitin adapter, Ufd1-Npl4. Notably, we show that, in cooperation with p97 and Ufd1-Npl4, SPRTN proteolyzed unfolded Eos-DPC, which relied on recognition of the DNA-crosslink by SPRTN. In a simplified unfolding assay, we further demonstrate that p97, while unfolding a protein substrate, can surmount the obstacle of a DNA crosslink site in the substrate. Thus, our data demonstrate that p97, in conjunction with Ufd1-Npl4, assists SPRTN-mediated proteolysis of tightly-folded proteins crosslinked to DNA, even threading bulky protein-DNA adducts. These findings will be relevant for understanding how cells handle DPCs to ensure genome stability and for designing strategies that target p97 in combination cancer therapy.

DNA-Protein Crosslink Proteolysis Repair.

Proteins that are covalently bound to DNA constitute a specific type of DNA lesion known as DNA-protein crosslinks (DPCs). DPCs represent physical obstacles to the progression of DNA replication. If not repaired, DPCs cause stalling of DNA replication forks that consequently leads to DNA double-strand breaks, the most cytotoxic DNA lesion. Although DPCs are common DNA lesions, the mechanism of DPC repair was unclear until now. Recent work unveiled that DPC repair is orchestrated by proteolysis performed by two distinct metalloproteases, SPARTAN in metazoans and Wss1 in yeast. This review summarizes recent discoveries on two proteases in DNA replication-coupled DPC repair and establishes DPC proteolysis repair as a separate DNA repair pathway for genome stability and protection from accelerated aging and cancer.

Wss1 homolog from Candida albicans and its role in DNA-protein crosslink tolerance.

Candida albicans is an opportunistic yeast that can cause life-threatening systemic infection in immunocompromised individuals. During infections, C. albicans has to cope with genotoxic stresses generated by the host immune system. DNA-protein crosslink (DPC), the covalent linkage of proteins with DNA, is one type of DNA damages that can be caused by the host immune response. DPCs are bulky lesions that interfere with the progression of replication and transcription machineries, and hence threaten genomic integrity. Accordingly, either a DPC tolerance mechanism or a DPC repair pathway is essential for C. albicans to maintain genomic stability and survive in the host. Here, we identified Wss1 (weak suppressor of Smt3) in C. albicans (CaWss1) using bioinformatics, genetic complementation, and biochemical studies. We showed that CaWss1 promotes cell survival under genotoxic stress conditions that generate DPCs and that the catalytic metalloprotease domain of CaWss1 is essential for its cellular function. Interactions of CaWss1 with Cdc48 and small ubiquitin-like modifier, although not strictly required, contribute to the function of CaWss1 in the suppression of the growth defects under DPC-inducing conditions. This report is the first investigation of the role of CaWss1 in DPC tolerance in C. albicans.

Emerging roles of Wss1 in the survival of Candida albicans under genotoxic stresses.

This perspective aims to discuss the potential physiological roles and regulation mechanisms of the recently identified Candida albicans Wss1 protease important in DNA-protein crosslink (DPC) tolerance and repair. DPC is a bulky DNA lesion that blocks essential DNA transactions; thus, it poses a significant threat to genome integrity if left unrepaired. Discoveries of Wss1 in Saccharomyces cerevisiae and SPRTN in human as DPC proteases have demonstrated the importance of protease function in DPC repair. Our recent study revealed that Wss1 in C. albicans, an opportunistic pathogen that can cause life-threatening infection in immunocompromised individuals, also promotes DPC tolerance similarly to both S. cerevisiae Wss1 and human SPRTN. However, its molecular mechanism and regulation are still poorly understood. Here, we briefly discuss the recent insights into C. albicans Wss1 based on the information from S. cerevisiae, as well as outline the aspect of this protein that could make it a potential target for antifungal drug development.

DNA-protein crosslink repair: proteases as DNA repair enzymes.

DNA-protein crosslinks (DPCs) are highly toxic DNA lesions because they interfere with DNA transactions. The recent discovery of a yeast protease that processes DPCs proteolytically raises the question whether DPC proteases also exist in higher eukaryotes. We argue here that the yeast enzyme, Wss1 (weak suppressor of smt3), is a member of a protease family whose mammalian representative is Spartan (SprT-like domain-containing protein)/DVC1 (DNA damage protein targeting VCP). DPC proteases may thus be common to all eukaryotes where they function as novel guardians of the genome.

SUMO orchestrates multiple alternative DNA-protein crosslink repair pathways.

Endogenous metabolites, environmental agents, and therapeutic drugs promote formation of covalent DNA-protein crosslinks (DPCs). Persistent DPCs compromise genome integrity and are eliminated by multiple repair pathways. Aberrant Top1-DNA crosslinks, or Top1ccs, are processed by Tdp1 and Wss1 functioning in parallel pathways in Saccharomyces cerevisiae. It remains obscure how cells choose between diverse mechanisms of DPC repair. Here, we show that several SUMO biogenesis factors (Ulp1, Siz2, Slx5, and Slx8) control repair of Top1cc or an analogous DPC lesion. Genetic analysis reveals that SUMO promotes Top1cc processing in the absence of Tdp1 but has an inhibitory role if cells additionally lack Wss1. In the tdp1Δ wss1Δ mutant, the E3 SUMO ligase Siz2 stimulates sumoylation in the vicinity of the DPC, but not SUMO conjugation to Top1. This Siz2-dependent sumoylation inhibits alternative DPC repair mechanisms, including Ddi1. Our findings suggest that SUMO tunes available repair pathways to facilitate faithful DPC repair.

Function and evolution of the DNA-protein crosslink proteases Wss1 and SPRTN.

Covalent DNA-protein crosslinks (DPCs) are highly toxic DNA adducts, which interfere with faithful DNA replication. The proteases Wss1 and SPRTN degrade DPCs and have emerged as crucially important DNA repair enzymes. Their protective role has been described in various model systems ranging from yeasts, plants, worms and flies to mice and humans. Loss of DPC proteases results in genome instability, cellular arrest, premature ageing and cancer predisposition. Here we discuss recent insights into the function and molecular mechanism of these enzymes. Furthermore, we present an in-depth phylogenetic analysis of the Wss1/SPRTN protease continuum. Remarkably flexible domain architectures and constantly changing protein-protein interaction motifs indicate ongoing evolutionary dynamics. Finally, we discuss recent data, which suggest that further partially-overlapping proteolytic systems targeting DPCs exist in eukaryotes. These new developments raise interesting questions regarding the division of labour between different DPC proteases and the mechanisms and principles of repair pathway choice.

Repair of DNA-protein crosslinks in plants.

DNA-protein crosslinks represent a severe kind of DNA damage as they disturb essential processes, such as transcription and DNA replication, due to their bulkiness. To ensure the maintenance of genome integrity, it is necessary for all living organisms to repair these lesions in a timely manner. Over recent years, much knowledge has been obtained regarding the repair of DNA-protein crosslinks (DPC), but it was only recently that the first insights into the mechanisms of DPC repair in plants were obtained. The plant DPC repair network consists of at least three parallel pathways that resolve DPC by distinct biochemical mechanisms. The endonuclease MUS81 resolves the DPC by cleaving the DNA part of the crosslink, the protease WSS1A is able to degrade the protein part and the tyrosyl-DNA-phosphodiesterase TDP1 can hydrolyse the crosslink between a protein and the DNA. However, due to the variety of different DPC types and the evolutionary conservation of pathways between eukaryotes, we expect that future research will reveal additional factors involved in DPC repair in plants.

Wss1 Promotes Replication Stress Tolerance by Degrading Histones.

Timely completion of DNA replication is central to accurate cell division and to the maintenance of genomic stability. However, certain DNA-protein interactions can physically impede DNA replication fork progression. Cells remove or bypass these physical impediments by different mechanisms to preserve DNA macromolecule integrity and genome stability. In Saccharomyces cerevisiae, Wss1, the DNA-protein crosslink repair protease, allows cells to tolerate hydroxyurea-induced replication stress, but the underlying mechanism by which Wss1 promotes this function has remained unknown. Here, we report that Wss1 provides cells tolerance to replication stress by directly degrading core histone subunits that non-specifically and non-covalently bind to single-stranded DNA. Unlike Wss1-dependent proteolysis of covalent DNA-protein crosslinks, proteolysis of histones does not require Cdc48 nor SUMO-binding activities. Wss1 thus acts as a multi-functional protease capable of targeting a broad range of covalent and non-covalent DNA-binding proteins to preserve genome stability during adverse conditions.

The yeast proteases Ddi1 and Wss1 are both involved in the DNA replication stress response.

Genome integrity and cell survival are dependent on proper replication stress response. Multiple repair pathways addressing obstacles generated by replication stress arose during evolution, and a detailed understanding of these processes is crucial for treatment of numerous human diseases. Here, we investigated the strong negative genetic interaction between two proteases involved in the DNA replication stress response, yeast Wss1 and Ddi1. While Wss1 proteolytically acts on DNA-protein crosslinks, mammalian DDI1 and DDI2 proteins remove RTF2 from stalled forks via a proposed proteasome shuttle hypothesis. We show that the double-deleted Δddi1, Δwss1 yeast strain is hypersensitive to the replication drug hydroxyurea and that this phenotype can be complemented only by catalytically competent Ddi1 protease. Furthermore, our data show the key involvement of the helical domain preceding the Ddi1 protease domain in response to replication stress caused by hydroxyurea, offering the first suggestion of this domain's biological function. Overall, our study provides a basis for a novel dual protease-based mechanism enabling yeast cells to counteract DNA replication stress.

A widely employed germ cell marker is an ancient disordered protein with reproductive functions in diverse eukaryotes.

The advent of sexual reproduction and the evolution of a dedicated germline in multicellular organisms are critical landmarks in eukaryotic evolution. We report an ancient family of GCNA (germ cell nuclear antigen) proteins that arose in the earliest eukaryotes, and feature a rapidly evolving intrinsically disordered region (IDR). Phylogenetic analysis reveals that GCNA proteins emerged before the major eukaryotic lineages diverged; GCNA predates the origin of a dedicated germline by a billion years. Gcna gene expression is enriched in reproductive cells across eukarya - either just prior to or during meiosis in single-celled eukaryotes, and in stem cells and germ cells of diverse multicellular animals. Studies of Gcna-mutant C. elegans and mice indicate that GCNA has functioned in reproduction for at least 600 million years. Homology to IDR-containing proteins implicated in DNA damage repair suggests that GCNA proteins may protect the genomic integrity of cells carrying a heritable genome.