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DNA–protein crosslink repair: proteases as DNA repair enzymes Julian Stingele1, Bianca Habermann2, and Stefan Jentsch1 1 2
Department of Molecular Cell Biology, Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany Computational Biology Group, Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany
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.
DNA–protein crosslinks Ensuring that genetic information is passed on faithfully to the next generation is essential for life, but this task is challenged by numerous assaults on the integrity of DNA. DNA lesions trigger mutagenesis and genome instability, and thus also contribute to tumorigenesis and aging. However, DNA repair pathways have evolved to counteract these threats. Because DNA lesions are very diverse in nature they require highly specific pathways for repair. Owing to intensive research during the past decades, dedicated repair factors for most types of DNA lesions have been discovered and the underlying mechanisms are now well understood. Surprisingly, however, one particular type of DNA lesion – covalent DNA–protein crosslinks (DPCs) – has been largely neglected even though DPCs are potentially very toxic. DPCs originate from either the permanent trapping of normally transient covalent protein–DNA intermediates during the reaction cycle of enzymes such as topoisomerases (‘enzymatic DPCs’), or from unspecific chemical crosslinking of proteins to DNA (‘nonenzymatic DPCs’), which are caused by agents such as ionizing radiation, UV light, or reactive aldehydes (Figure 1A). Notably, reactive aldehydes are produced metabolically (e.g., during the oxidation of ethanol to acetaldehyde by alcohol dehydrogenases) and even directly at chromatin, where histone demethylation produces formaldehyde [1]. The toxicity of aldehyde-induced DNA lesions is strikingly exemplified by the finding that mice deficient for the acetaldehyde-detoxifying enzyme aldehyde dehydrogenase 2 (ALDH2) develop Corresponding author: Jentsch, S. ([email protected]). Keywords: DNA–protein crosslinks; DNA repair; DPC protease; DVC1; Spartan; SPRTN; Wss1. 0968-0004/ ß 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tibs.2014.10.012
leukemia and anemia if the animals are additionally defective in functional DNA repair pathways [2,3]. DPCs are particularly toxic because they interfere with essential DNA transactions. For example, DPCs inhibit unwinding of the DNA duplex by replicative helicases, thus prohibiting replication completion and consequently cell division (Figure 1B). However, whether cells possess DPC-specific repair mechanisms remained unclear until recently. DNA–protein crosslink repair Nucleotide excision repair (NER) provides resistance towards formaldehyde and is responsible for removing the majority of formaldehyde-induced DPCs in yeast [4,5]. However, DPCs that are resistant to NER or have escaped repair before S-phase are especially life-threatening because they will likely stall approaching replication forks. Recent genetic data obtained in yeast indicate that DPC-mediated replication stalling is countered by two distinct but partially redundant mechanisms: DPC repair and DPC tolerance (Figure 1C). The DPC repair pathway is built around the DPC-processing protease (termed DPC protease here) Wss1, which provides resistance towards topoisomerase 1 (Top1)- and formaldehyde-induced DPCs [5]. The current model is that Wss1 proteolytically breaks down the bulk of the protein components of DPCs, thereby enabling progression of the replicative helicase. The remaining peptide remnant covalently bound to DNA will still block replicative polymerases, but replication can continue upon recruitment of mutagenic (because they potentially incorporate wrong nucleotides) translesion synthesis (TLS) polymerases which are able to replicate even across bulky DNA lesions. This model is supported by the observation that formaldehyde induces TLS-mediated mutagenesis, which partially depends on Wss1 [5]. DPC tolerance, the alternative pathway to DPC repair, may allow replication completion but does not remove the DPCs. DPC tolerance likely involves the generation of single-ended DNA double-strand breaks (DSBs) either by replication fork run-off, as in the case of Top1-dependent DPCs, or endonucleolytic cleavage, as for nonenzymatic DPCs. The DSBs are then subsequently repaired by break-induced replication (BIR) or homologous recombination (HR), which risk genome rearrangements. Because this alternative pathway is principally active in the absence of the DPC protease, cells lacking Wss1 suffer from hyper-recombination and genomic rearrangements [5]. Thus, DPC proteases are highly advantageous for cells because they enable replication completion in the face of DPCs and concurrently promote genome stability. Intriguingly, Trends in Biochemical Sciences, February 2015, Vol. 40, No. 2
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Figure 1. Causes, problems, and solutions for DNA–protein crosslinks (DPCs). (A) Sources of DPCs. Enzymatic DPCs are caused by trapping of normally transient covalent enzyme–DNA reaction intermediates. Topoisomerase 1 (Top1) can be trapped if nearby DNA damage (such as abasic sites) inhibits completion of the enzymatic reaction cycle. Top1-trapping can also be induced by small molecules such as camptothecin, which intercalates within the enzyme–DNA interface. Nonenzymatic DPCs are caused by nonspecific chemical crosslinking of proteins to DNA by agents originating from endogenous and exogenous sources. Reactive aldehydes, for example acetaldehyde and formaldehyde, are produced metabolically from ethanol oxidation or histone demethylation, respectively. Exogenous agents causing DPCs include ionizing radiation, UV light, and chemical crosslinkers such as platinum-based anticancer drugs (e.g., cisplatin). (B) DPCs may cause stalling of approaching replication forks, especially when located on the leading strand. Persistent stalling of replication forks inhibits replication completion, and consequently cell division, and potentially causes cell death. (C) Solutions for coping with DPCs. Cells address the problem caused by DPC-stalled replication forks by two distinct but partially redundant mechanisms. The current model for DPC repair in yeast involves proteolytic breakdown of the protein component of the DPC by the DPC protease Wss1. DPC proteolysis allows progression of the replicative helicase. However, peptide remnants remaining crosslinked to DNA may require translesion synthesis (TLS) polymerases for replication completion, potentially causing mutagenesis. Particularly when cells are deficient in DPC repair, DPC tolerance via a recombination-based mechanism may occur. Persistent fork stalling may cause fork collapse and cleavage by endonuclease, leading to the generation of a single-ended double-strand break (DSB), which can be repaired by break-induced replication (BIR) or homologous recombination (HR). DPC tolerance pathways will leave the DPCs unrepaired and bear the danger of inducing genome rearrangements and instability.
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Forum DPC repair was very recently shown to operate in Xenopus laevis egg extracts as well, where DPC-containing plasmids are repaired by a mechanism strikingly similar to DPC repair in yeast [6]. However, the identity of the involved DPC protease remained unclear. A conserved family of DPC proteases? To identify proteins homologous to Wss1 in other species, we conducted reciprocal BLAST searches. Proteins with high sequence similarity to Saccharomyces cerevisiae Wss1 (termed the ‘Wss1 branch’) can be readily identified in various other fungi and in plants (Figure 2A). Plants and some fungi additionally express a second class of Wss1-like proteins that are characterized by an N-terminal ubiquitinlike (UBL) domain (termed the ‘UBL-Wss1 branch’). Metazoans, by contrast, possess proteins significantly more divergent from Wss1, but nevertheless with meaningful homology to the protease domain of Wss1. Notably, the human member of this class (termed the ‘Spartan branch’) is Spartan (SPRTN, also known as DVC1 or C1orf124), a protein previously speculated to be potentially related to Wss1 [7]. A detailed phylogenetic analysis based on sequence similarity and domain organization indeed indicates a common ancestry for Wss1 and Spartan. In addition to conserved N-terminal protease domains, Wss1 and Spartan both possess C-terminal tail regions harboring various protein–protein interaction modules (Figure 2B). Notably, both proteins harbor sequence motifs for binding to the segregase Cdc48 (termed p97 in higher eukaryotes), and binding to this chaperone-like enzyme has been confirmed for yeast Wss1 and human Spartan [5,7–9]. In addition, Spartan and Wss1 possess near their C-termini interaction domains for either ubiquitin or small ubiquitin-like modifier (SUMO), respectively. Notably, proteins of the Spartan branch additionally possess short interaction motifs (PIP-boxes) for interaction with the replication clamp PCNA (proliferating cell nuclear antigen). In addition to these structural similarities, orthology between Wss1 and Spartan is further indicated by functional data (Figure 2C). Importantly, both Wss1 and Spartan are believed to function in DNA repair coupled to DNA replication. A series of recent reports have described the involvement of Spartan in translesion synthesis, albeit with conflicting results and interpretations [7–13]. Some groups report that Spartan recruitment to stalled replication forks depends on DNA damage-induced PCNA ubiquitylation by the Rad18 ubiquitin ligase [9–11,13]; by contrast, other reports suggest that Spartan recruitment is independent of this PCNA modification [7,8]. Furthermore, it is also under debate whether Spartan promotes DNA damage-induced Polh (TLS polymerase) foci formation [10,11] or, conversely, disassembles them [7,8]. However, despite these mechanistic inconsistencies, all reports agree that Spartan acts at replication forks, as was also suggested for Wss1 [5]. An additional shared feature of Wss1 and Spartan is that both proteins bind the segregase Cdc48 (p97), and this physical interaction is essential for their function in vivo [5,9]. Moreover, both Wss1 and Spartan are targeted to DNA – either directly via a DNA-binding domain (Wss1), or indirectly through interaction with DNA-bound PCNA (Spartan) [5,10].
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Spartan does not seem to possess a Wss1-like DNA-binding domain, but direct DNA binding, possibly through other protein domains, has not been explored. Interestingly, both proteases bind to ubiquitin family proteins, and this feature might be used to target the proteins to their sites of action. Indeed, Spartan variants deficient in ubiquitin binding (UBZ mutant variants) fail to localize to sites of DNA damage and are unable to complement Spartan deficiency [10]. Similarly, Wss1 variants lacking SUMO binding properties (SIM mutant variants) fail to fully complement phenotypes linked to Wss1 deficiency [5]. The identity of the SUMOylated partner of Wss1 is currently unknown, but for Spartan some reports suggest that ubiquitylated PCNA might be the crucial target. Restraining protease activities is crucial to avoid unwanted and potentially deleterious turnover of cellular proteins. Typical measures are the expression of the proteases as inactive precursors (zymogens), spatial sequestration of the enzymes (lysosomal proteases), compartmentalization of the active sites (proteasomes), or the use of active sites with high sequence specificity. Regulation of Wss1 needs to be exceptionally tight given that the enzyme has the astonishing ability to digest any DNA-bound protein in vitro irrespective of its identity [5]. In addition to perhaps restraining Wss1 activity via specific targeting, Wss1 might be controlled by protein turnover. Indeed, Wss1 is expressed at extremely low levels, and also undergoes self-cleavage in trans (i.e., one Wss1 protein cleaves another) [5], a feature possibly used for enzyme inactivation. Spartan is instead proteasomally degraded through a mechanism involving the cell cycle regulated E3-ligase anaphase-promoting complex – Cdc20 homolog 1 (APC-Cdh1) [7]. By these means, Spartan activity is restricted to the S/G2-phase of the cell cycle. How the proteolytic activity of Spartan might be contained is unknown because the function of its protease domain has only been sparsely investigated. Currently only one report directly addressed the possible proteolytic activity of Spartan, indicating that its protease activity is needed for mutagenesis suppression [12]. Other recent work did not examine the protease domain, and described Spartan as a scaffold rather than an enzyme [7–11]. Because of this knowledge gap regarding the proteolytic activity of Spartan and, given the current inconsistencies related to the potential role of Spartan in TLS, further efforts will be necessary to clarify its role in DNA repair. An idea of how far-reaching the cellular activity of Spartan is came from recent studies on flies [14]. In Drosophila, Spartan deficiency results in a specific failure to replicate paternal DNA during the first mitosis of the zygote. The authors speculate that the dense packaging of paternal DNA in sperms by topoisomerases might cause DNA damage, perhaps DPC formation, thus requiring Spartan for repair. Because of the striking similarities between Wss1 and Spartan discussed here, we would like to propose that higher eukaryotes possess DPC proteases and that Spartan may fulfill Wss1-related DPC repair functions in humans. Given the known function of Wss1 for genome stability in yeast, it was not surprising that Spartan was very recently shown to be required for tumor suppression in humans [15]. Strikingly, patients with mutations in the 69
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Figure 2. A conserved family of DPC proteases? (A) Phylogenetic tree of Wss1 and Spartan proteases. Reciprocal best-hit relationships as well as common domain structures are strong evidence for phylogenetic conservation of Wss1/Spartan proteases in eukaryotes. The protease domain is well conserved in fungi and plants (Wss1 branch, bottom of the tree) and is more divergent in metazoans (Spartan branch, top of the tree). Paralogs of Wss1 bearing a ubiquitin-like (UBL) domain exist in plants as well as in some fungal species (UBL-Wss1 branch). Dots indicate stable branches (bootstrap value 80), the scale bar indicates substitutions per site. Species abbreviations, accession numbers, as well as the multiple sequence alignment used to generate the phylogenetic tree are available in the supplementary material online. (B) Domain structure of Wss1 and Spartan proteins. Wss1 and Spartan branches share similar functional motifs. Indicated domains are the conserved core of the protease domain (yellow stripe denotes the position of the active site), Cdc48/p97 (SHP-box, VIM, PUB), ubiquitin (UBZ), SUMO (SIM) and PCNA (PIP-box) binding motifs and ubiquitin-like domains (UBL). Annotated multiple sequence alignments of species groups are available in the supplementary material online. (C) Wss1 and Spartan share common regulatory principles. Both Wss1 (red) and Spartan (blue) require binding to the segregase Cdc48 (p97 in mammals) to perform their cellular functions. Whereas Wss1 is able to bind DNA directly, Spartan is recruited to DNA via interaction with the replication clamp PCNA. In addition, both proteases bind to ubiquitin or ubiquitin-like modifications via their C-terminal tails. Wss1, which is expressed at extremely low levels, is additionally regulated via self-cleavage. Conversely, Spartan levels are regulated by cell cycle-specific proteasomal degradation.
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Forum gene coding for Spartan developed early-onset hepatocellular carcinomas and displayed a premature aging syndrome. Thus, DPC repair appears to be crucial for genome integrity in humans and consequently for preventing tumorigenesis. Acknowledgments We thank Kay Hofmann for sharing unpublished information. S.J. is supported by the Max Planck Society, the Deutsche Forschungsgemeinschaft, the Center for Integrated Protein Science Munich, a European Research Council (ERC) Advanced Grant, and the LouisJeantet Foundation.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.tibs.2014.10.012.
References 1 Barker, S. et al. (2005) DNA–protein crosslinks: their induction, repair, and biological consequences. Mutat. Res. 589, 111–135 2 Langevin, F. et al. (2011) Fancd2 counteracts the toxic effects of naturally produced aldehydes in mice. Nature 475, 53–58 3 Garaycoechea, J.I. et al. (2012) Genotoxic consequences of endogenous aldehydes on mouse haematopoietic stem cell function. Nature 489, 571–575 4 de Graaf, B. et al. (2009) Cellular pathways for DNA repair and damage tolerance of formaldehyde-induced DNA–protein crosslinks. DNA Repair 8, 1207–1214
Trends in Biochemical Sciences February 2015, Vol. 40, No. 2 5 Stingele, J. et al. (2014) A DNA-dependent protease involved in DNA– protein crosslink repair. Cell 158, 327–338 6 Duxin, J.P. et al. (2014) Repair of a DNA–protein crosslink by replication-coupled proteolysis. Cell 159, 346–357 7 Mosbech, A. et al. (2012) DVC1 (C1orf124) is a DNA damage-targeting p97 adaptor that promotes ubiquitin-dependent responses to replication blocks. Nat. Struct. Mol. Biol. 19, 1084–1092 8 Davis, E.J. et al. (2012) DVC1 (C1orf124) recruits the p97 protein segregase to sites of DNA damage. Nat. Struct. Mol. Biol. 19, 1093–1100 9 Ghosal, G. et al. (2012) Proliferating cell nuclear antigen (PCNA)binding protein C1orf124 is a regulator of translesion synthesis. J. Biol. Chem. 287, 34225–34233 10 Centore, R.C. et al. (2012) Spartan/C1orf124, a reader of PCNA ubiquitylation and a regulator of UV-induced DNA damage response. Mol. Cell 46, 625–635 11 Juhasz, S. et al. (2012) Characterization of human Spartan/C1orf124, an ubiquitin-PCNA interacting regulator of DNA damage tolerance. Nucleic Acids Res. 40, 10795–10808 12 Kim, M.S. et al. (2013) Regulation of error-prone translesion synthesis by Spartan/C1orf124. Nucleic Acids Res. 41, 1661–1668 13 Machida, Y. et al. (2012) Spartan/C1orf124 is important to prevent UVinduced mutagenesis. Cell Cycle 11, 3395–3402 14 Delabaere, L. et al. (2014) The Spartan ortholog maternal haploid is required for paternal chromosome integrity in the Drosophila zygote. Curr. Biol. 24, 2281–2287 15 Lessel, D. et al. (2014) Mutations in SPRTN cause early onset hepatocellular carcinoma, genomic instability and progeroid features. Nat. Genet. 46, 1239–1244
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DNA–protein crosslink repair: proteases as DNA repair enzymes Julian Stingele1, Bianca Habermann2, and Stefan Jentsch1 1 2
Department of Molecular Cell Biology, Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany Computational Biology Group, Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany
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.
DNA–protein crosslinks Ensuring that genetic information is passed on faithfully to the next generation is essential for life, but this task is challenged by numerous assaults on the integrity of DNA. DNA lesions trigger mutagenesis and genome instability, and thus also contribute to tumorigenesis and aging. However, DNA repair pathways have evolved to counteract these threats. Because DNA lesions are very diverse in nature they require highly specific pathways for repair. Owing to intensive research during the past decades, dedicated repair factors for most types of DNA lesions have been discovered and the underlying mechanisms are now well understood. Surprisingly, however, one particular type of DNA lesion – covalent DNA–protein crosslinks (DPCs) – has been largely neglected even though DPCs are potentially very toxic. DPCs originate from either the permanent trapping of normally transient covalent protein–DNA intermediates during the reaction cycle of enzymes such as topoisomerases (‘enzymatic DPCs’), or from unspecific chemical crosslinking of proteins to DNA (‘nonenzymatic DPCs’), which are caused by agents such as ionizing radiation, UV light, or reactive aldehydes (Figure 1A). Notably, reactive aldehydes are produced metabolically (e.g., during the oxidation of ethanol to acetaldehyde by alcohol dehydrogenases) and even directly at chromatin, where histone demethylation produces formaldehyde [1]. The toxicity of aldehyde-induced DNA lesions is strikingly exemplified by the finding that mice deficient for the acetaldehyde-detoxifying enzyme aldehyde dehydrogenase 2 (ALDH2) develop Corresponding author: Jentsch, S. ([email protected]). Keywords: DNA–protein crosslinks; DNA repair; DPC protease; DVC1; Spartan; SPRTN; Wss1. 0968-0004/ ß 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tibs.2014.10.012
leukemia and anemia if the animals are additionally defective in functional DNA repair pathways [2,3]. DPCs are particularly toxic because they interfere with essential DNA transactions. For example, DPCs inhibit unwinding of the DNA duplex by replicative helicases, thus prohibiting replication completion and consequently cell division (Figure 1B). However, whether cells possess DPC-specific repair mechanisms remained unclear until recently. DNA–protein crosslink repair Nucleotide excision repair (NER) provides resistance towards formaldehyde and is responsible for removing the majority of formaldehyde-induced DPCs in yeast [4,5]. However, DPCs that are resistant to NER or have escaped repair before S-phase are especially life-threatening because they will likely stall approaching replication forks. Recent genetic data obtained in yeast indicate that DPC-mediated replication stalling is countered by two distinct but partially redundant mechanisms: DPC repair and DPC tolerance (Figure 1C). The DPC repair pathway is built around the DPC-processing protease (termed DPC protease here) Wss1, which provides resistance towards topoisomerase 1 (Top1)- and formaldehyde-induced DPCs [5]. The current model is that Wss1 proteolytically breaks down the bulk of the protein components of DPCs, thereby enabling progression of the replicative helicase. The remaining peptide remnant covalently bound to DNA will still block replicative polymerases, but replication can continue upon recruitment of mutagenic (because they potentially incorporate wrong nucleotides) translesion synthesis (TLS) polymerases which are able to replicate even across bulky DNA lesions. This model is supported by the observation that formaldehyde induces TLS-mediated mutagenesis, which partially depends on Wss1 [5]. DPC tolerance, the alternative pathway to DPC repair, may allow replication completion but does not remove the DPCs. DPC tolerance likely involves the generation of single-ended DNA double-strand breaks (DSBs) either by replication fork run-off, as in the case of Top1-dependent DPCs, or endonucleolytic cleavage, as for nonenzymatic DPCs. The DSBs are then subsequently repaired by break-induced replication (BIR) or homologous recombination (HR), which risk genome rearrangements. Because this alternative pathway is principally active in the absence of the DPC protease, cells lacking Wss1 suffer from hyper-recombination and genomic rearrangements [5]. Thus, DPC proteases are highly advantageous for cells because they enable replication completion in the face of DPCs and concurrently promote genome stability. Intriguingly, Trends in Biochemical Sciences, February 2015, Vol. 40, No. 2
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Figure 1. Causes, problems, and solutions for DNA–protein crosslinks (DPCs). (A) Sources of DPCs. Enzymatic DPCs are caused by trapping of normally transient covalent enzyme–DNA reaction intermediates. Topoisomerase 1 (Top1) can be trapped if nearby DNA damage (such as abasic sites) inhibits completion of the enzymatic reaction cycle. Top1-trapping can also be induced by small molecules such as camptothecin, which intercalates within the enzyme–DNA interface. Nonenzymatic DPCs are caused by nonspecific chemical crosslinking of proteins to DNA by agents originating from endogenous and exogenous sources. Reactive aldehydes, for example acetaldehyde and formaldehyde, are produced metabolically from ethanol oxidation or histone demethylation, respectively. Exogenous agents causing DPCs include ionizing radiation, UV light, and chemical crosslinkers such as platinum-based anticancer drugs (e.g., cisplatin). (B) DPCs may cause stalling of approaching replication forks, especially when located on the leading strand. Persistent stalling of replication forks inhibits replication completion, and consequently cell division, and potentially causes cell death. (C) Solutions for coping with DPCs. Cells address the problem caused by DPC-stalled replication forks by two distinct but partially redundant mechanisms. The current model for DPC repair in yeast involves proteolytic breakdown of the protein component of the DPC by the DPC protease Wss1. DPC proteolysis allows progression of the replicative helicase. However, peptide remnants remaining crosslinked to DNA may require translesion synthesis (TLS) polymerases for replication completion, potentially causing mutagenesis. Particularly when cells are deficient in DPC repair, DPC tolerance via a recombination-based mechanism may occur. Persistent fork stalling may cause fork collapse and cleavage by endonuclease, leading to the generation of a single-ended double-strand break (DSB), which can be repaired by break-induced replication (BIR) or homologous recombination (HR). DPC tolerance pathways will leave the DPCs unrepaired and bear the danger of inducing genome rearrangements and instability.
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Forum DPC repair was very recently shown to operate in Xenopus laevis egg extracts as well, where DPC-containing plasmids are repaired by a mechanism strikingly similar to DPC repair in yeast [6]. However, the identity of the involved DPC protease remained unclear. A conserved family of DPC proteases? To identify proteins homologous to Wss1 in other species, we conducted reciprocal BLAST searches. Proteins with high sequence similarity to Saccharomyces cerevisiae Wss1 (termed the ‘Wss1 branch’) can be readily identified in various other fungi and in plants (Figure 2A). Plants and some fungi additionally express a second class of Wss1-like proteins that are characterized by an N-terminal ubiquitinlike (UBL) domain (termed the ‘UBL-Wss1 branch’). Metazoans, by contrast, possess proteins significantly more divergent from Wss1, but nevertheless with meaningful homology to the protease domain of Wss1. Notably, the human member of this class (termed the ‘Spartan branch’) is Spartan (SPRTN, also known as DVC1 or C1orf124), a protein previously speculated to be potentially related to Wss1 [7]. A detailed phylogenetic analysis based on sequence similarity and domain organization indeed indicates a common ancestry for Wss1 and Spartan. In addition to conserved N-terminal protease domains, Wss1 and Spartan both possess C-terminal tail regions harboring various protein–protein interaction modules (Figure 2B). Notably, both proteins harbor sequence motifs for binding to the segregase Cdc48 (termed p97 in higher eukaryotes), and binding to this chaperone-like enzyme has been confirmed for yeast Wss1 and human Spartan [5,7–9]. In addition, Spartan and Wss1 possess near their C-termini interaction domains for either ubiquitin or small ubiquitin-like modifier (SUMO), respectively. Notably, proteins of the Spartan branch additionally possess short interaction motifs (PIP-boxes) for interaction with the replication clamp PCNA (proliferating cell nuclear antigen). In addition to these structural similarities, orthology between Wss1 and Spartan is further indicated by functional data (Figure 2C). Importantly, both Wss1 and Spartan are believed to function in DNA repair coupled to DNA replication. A series of recent reports have described the involvement of Spartan in translesion synthesis, albeit with conflicting results and interpretations [7–13]. Some groups report that Spartan recruitment to stalled replication forks depends on DNA damage-induced PCNA ubiquitylation by the Rad18 ubiquitin ligase [9–11,13]; by contrast, other reports suggest that Spartan recruitment is independent of this PCNA modification [7,8]. Furthermore, it is also under debate whether Spartan promotes DNA damage-induced Polh (TLS polymerase) foci formation [10,11] or, conversely, disassembles them [7,8]. However, despite these mechanistic inconsistencies, all reports agree that Spartan acts at replication forks, as was also suggested for Wss1 [5]. An additional shared feature of Wss1 and Spartan is that both proteins bind the segregase Cdc48 (p97), and this physical interaction is essential for their function in vivo [5,9]. Moreover, both Wss1 and Spartan are targeted to DNA – either directly via a DNA-binding domain (Wss1), or indirectly through interaction with DNA-bound PCNA (Spartan) [5,10].
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Spartan does not seem to possess a Wss1-like DNA-binding domain, but direct DNA binding, possibly through other protein domains, has not been explored. Interestingly, both proteases bind to ubiquitin family proteins, and this feature might be used to target the proteins to their sites of action. Indeed, Spartan variants deficient in ubiquitin binding (UBZ mutant variants) fail to localize to sites of DNA damage and are unable to complement Spartan deficiency [10]. Similarly, Wss1 variants lacking SUMO binding properties (SIM mutant variants) fail to fully complement phenotypes linked to Wss1 deficiency [5]. The identity of the SUMOylated partner of Wss1 is currently unknown, but for Spartan some reports suggest that ubiquitylated PCNA might be the crucial target. Restraining protease activities is crucial to avoid unwanted and potentially deleterious turnover of cellular proteins. Typical measures are the expression of the proteases as inactive precursors (zymogens), spatial sequestration of the enzymes (lysosomal proteases), compartmentalization of the active sites (proteasomes), or the use of active sites with high sequence specificity. Regulation of Wss1 needs to be exceptionally tight given that the enzyme has the astonishing ability to digest any DNA-bound protein in vitro irrespective of its identity [5]. In addition to perhaps restraining Wss1 activity via specific targeting, Wss1 might be controlled by protein turnover. Indeed, Wss1 is expressed at extremely low levels, and also undergoes self-cleavage in trans (i.e., one Wss1 protein cleaves another) [5], a feature possibly used for enzyme inactivation. Spartan is instead proteasomally degraded through a mechanism involving the cell cycle regulated E3-ligase anaphase-promoting complex – Cdc20 homolog 1 (APC-Cdh1) [7]. By these means, Spartan activity is restricted to the S/G2-phase of the cell cycle. How the proteolytic activity of Spartan might be contained is unknown because the function of its protease domain has only been sparsely investigated. Currently only one report directly addressed the possible proteolytic activity of Spartan, indicating that its protease activity is needed for mutagenesis suppression [12]. Other recent work did not examine the protease domain, and described Spartan as a scaffold rather than an enzyme [7–11]. Because of this knowledge gap regarding the proteolytic activity of Spartan and, given the current inconsistencies related to the potential role of Spartan in TLS, further efforts will be necessary to clarify its role in DNA repair. An idea of how far-reaching the cellular activity of Spartan is came from recent studies on flies [14]. In Drosophila, Spartan deficiency results in a specific failure to replicate paternal DNA during the first mitosis of the zygote. The authors speculate that the dense packaging of paternal DNA in sperms by topoisomerases might cause DNA damage, perhaps DPC formation, thus requiring Spartan for repair. Because of the striking similarities between Wss1 and Spartan discussed here, we would like to propose that higher eukaryotes possess DPC proteases and that Spartan may fulfill Wss1-related DPC repair functions in humans. Given the known function of Wss1 for genome stability in yeast, it was not surprising that Spartan was very recently shown to be required for tumor suppression in humans [15]. Strikingly, patients with mutations in the 69
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Trends in Biochemical Sciences February 2015, Vol. 40, No. 2
Figure 2. A conserved family of DPC proteases? (A) Phylogenetic tree of Wss1 and Spartan proteases. Reciprocal best-hit relationships as well as common domain structures are strong evidence for phylogenetic conservation of Wss1/Spartan proteases in eukaryotes. The protease domain is well conserved in fungi and plants (Wss1 branch, bottom of the tree) and is more divergent in metazoans (Spartan branch, top of the tree). Paralogs of Wss1 bearing a ubiquitin-like (UBL) domain exist in plants as well as in some fungal species (UBL-Wss1 branch). Dots indicate stable branches (bootstrap value 80), the scale bar indicates substitutions per site. Species abbreviations, accession numbers, as well as the multiple sequence alignment used to generate the phylogenetic tree are available in the supplementary material online. (B) Domain structure of Wss1 and Spartan proteins. Wss1 and Spartan branches share similar functional motifs. Indicated domains are the conserved core of the protease domain (yellow stripe denotes the position of the active site), Cdc48/p97 (SHP-box, VIM, PUB), ubiquitin (UBZ), SUMO (SIM) and PCNA (PIP-box) binding motifs and ubiquitin-like domains (UBL). Annotated multiple sequence alignments of species groups are available in the supplementary material online. (C) Wss1 and Spartan share common regulatory principles. Both Wss1 (red) and Spartan (blue) require binding to the segregase Cdc48 (p97 in mammals) to perform their cellular functions. Whereas Wss1 is able to bind DNA directly, Spartan is recruited to DNA via interaction with the replication clamp PCNA. In addition, both proteases bind to ubiquitin or ubiquitin-like modifications via their C-terminal tails. Wss1, which is expressed at extremely low levels, is additionally regulated via self-cleavage. Conversely, Spartan levels are regulated by cell cycle-specific proteasomal degradation.
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Forum gene coding for Spartan developed early-onset hepatocellular carcinomas and displayed a premature aging syndrome. Thus, DPC repair appears to be crucial for genome integrity in humans and consequently for preventing tumorigenesis. Acknowledgments We thank Kay Hofmann for sharing unpublished information. S.J. is supported by the Max Planck Society, the Deutsche Forschungsgemeinschaft, the Center for Integrated Protein Science Munich, a European Research Council (ERC) Advanced Grant, and the LouisJeantet Foundation.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.tibs.2014.10.012.
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