DNA-Protein Crosslinks and Their Resolution.

Covalent DNA-protein crosslinks (DPCs) are pervasive DNA lesions that interfere with essential chromatin processes such as transcription or replication. This review strives to provide an overview of the sources and principles of cellular DPC formation. DPCs are caused by endogenous reactive metabolites and various chemotherapeutic agents. However, in certain conditions DPCs also arise physiologically in cells. We discuss the cellular mechanisms resolving these threats to genomic integrity. Detection and repair of DPCs require not only the action of canonical DNA repair pathways but also the activity of specialized proteolytic enzymes-including proteases of the SPRTN/Wss1 family-to degrade the crosslinked protein. Loss of DPC repair capacity has dramatic consequences, ranging from genome instability in yeast and worms to cancer predisposition and premature aging in mice and humans.

The FANCJ helicase unfolds DNA-protein crosslinks to promote their repair.

Endogenous and exogenous agents generate DNA-protein crosslinks (DPCs), whose replication-dependent degradation by the SPRTN protease suppresses aging and liver cancer. SPRTN is activated after the replicative CMG helicase bypasses a DPC and polymerase extends the nascent strand to the adduct. Here, we identify a role for the 5'-to-3' helicase FANCJ in DPC repair. In addition to supporting CMG bypass, FANCJ is essential for SPRTN activation. FANCJ binds ssDNA downstream of the DPC and uses its ATPase activity to unfold the protein adduct, which exposes the underlying DNA and enables cleavage of the adduct. FANCJ-dependent DPC unfolding is also essential for translesion DNA synthesis past DPCs that cannot be degraded. In summary, our results show that helicase-mediated protein unfolding enables multiple events in DPC repair.

RNF14-dependent atypical ubiquitylation promotes translation-coupled resolution of RNA-protein crosslinks.

Reactive aldehydes are abundant endogenous metabolites that challenge homeostasis by crosslinking cellular macromolecules. Aldehyde-induced DNA damage requires repair to prevent cancer and premature aging, but it is unknown whether cells also possess mechanisms that resolve aldehyde-induced RNA lesions. Here, we establish photoactivatable ribonucleoside-enhanced crosslinking (PAR-CL) as a model system to study RNA crosslinking damage in the absence of confounding DNA damage in human cells. We find that such RNA damage causes translation stress by stalling elongating ribosomes, which leads to collisions with trailing ribosomes and activation of multiple stress response pathways. Moreover, we discovered a translation-coupled quality control mechanism that resolves covalent RNA-protein crosslinks. Collisions between translating ribosomes and crosslinked mRNA-binding proteins trigger their modification with atypical K6- and K48-linked ubiquitin chains. Ubiquitylation requires the E3 ligase RNF14 and leads to proteasomal degradation of the protein adduct. Our findings identify RNA lesion-induced translational stress as a central component of crosslinking damage.

Releasing the trap: How the segregase p97 extracts PARP1 from chromatin.

Krastev et al. (2022) identify a cellular mechanism that counteracts cytotoxic trapping of PARP1 induced by clinical PARP inhibitors. SUMO-targeted ubiquitylation of trapped PARP1 is shown to trigger the enzymes' extraction from chromatin by the p97 ATPase.

Mechanisms of DNA-protein crosslink repair.

Covalent DNA-protein crosslinks (DPCs, also known as protein adducts) of topoisomerases and other proteins with DNA are highly toxic DNA lesions. Of note, chemical agents that induce DPCs include widely used classes of chemotherapeutics. Their bulkiness blocks virtually every chromatin-based process and makes them intractable for repair by canonical repair pathways. Distinct DPC repair pathways employ unique points of attack and are crucial for the maintenance of genome stability. Tyrosyl-DNA phosphodiesterases (TDPs) directly hydrolyse the covalent linkage between protein and DNA. The MRE11-RAD50-NBS1 (MRN) nuclease complex targets the DNA component of DPCs, excising the fragment affected by the lesion, whereas proteases of the spartan (SPRTN)/weak suppressor of SMT3 protein 1 (Wss1) family target the protein component. Loss of these pathways renders cells sensitive to DPC-inducing chemotherapeutics, and DPC repair pathways are thus attractive targets for combination cancer therapy.

A DNA-dependent protease involved in DNA-protein crosslink repair.

Toxic DNA-protein crosslinks (DPCs) arise by ionizing irradiation and UV light, are particularly caused by endogenously produced reactive compounds such as formaldehyde, and also occur during compromised topoisomerase action. Although nucleotide excision repair and homologous recombination contribute to cell survival upon DPCs, hardly anything is known about mechanisms that target the protein component of DPCs directly. Here, we identify the metalloprotease Wss1 as being crucial for cell survival upon exposure to formaldehyde and topoisomerase 1-dependent DNA damage. Yeast mutants lacking Wss1 accumulate DPCs and exhibit gross chromosomal rearrangements. Notably, in vitro assays indicate that substrates such as topoisomerase 1 are processed by the metalloprotease directly and in a DNA-dependent manner. Thus, our data suggest that Wss1 contributes to survival of DPC-harboring cells by acting on DPCs proteolytically. We propose that DPC proteolysis enables repair of these unique lesions via downstream canonical DNA repair pathways.

Decitabine cytotoxicity is promoted by dCMP deaminase DCTD and mitigated by SUMO-dependent E3 ligase TOPORS.

The nucleoside analogue decitabine (or 5-aza-dC) is used to treat several haematological cancers. Upon its triphosphorylation and incorporation into DNA, 5-aza-dC induces covalent DNA methyltransferase 1 DNA-protein crosslinks (DNMT1-DPCs), leading to DNA hypomethylation. However, 5-aza-dC's clinical outcomes vary, and relapse is common. Using genome-scale CRISPR/Cas9 screens, we map factors determining 5-aza-dC sensitivity. Unexpectedly, we find that loss of the dCMP deaminase DCTD causes 5-aza-dC resistance, suggesting that 5-aza-dUMP generation is cytotoxic. Combining results from a subsequent genetic screen in DCTD-deficient cells with the identification of the DNMT1-DPC-proximal proteome, we uncover the ubiquitin and SUMO1 E3 ligase, TOPORS, as a new DPC repair factor. TOPORS is recruited to SUMOylated DNMT1-DPCs and promotes their degradation. Our study suggests that 5-aza-dC-induced DPCs cause cytotoxicity when DPC repair is compromised, while cytotoxicity in wild-type cells arises from perturbed nucleotide metabolism, potentially laying the foundations for future identification of predictive biomarkers for decitabine treatment.

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.

A non-proteolytic release mechanism for HMCES-DNA-protein crosslinks.

The conserved protein HMCES crosslinks to abasic (AP) sites in ssDNA to prevent strand scission and the formation of toxic dsDNA breaks during replication. Here, we report a non-proteolytic release mechanism for HMCES-DNA-protein crosslinks (DPCs), which is regulated by DNA context. In ssDNA and at ssDNA-dsDNA junctions, HMCES-DPCs are stable, which efficiently protects AP sites against spontaneous incisions or cleavage by APE1 endonuclease. In contrast, HMCES-DPCs are released in dsDNA, allowing APE1 to initiate downstream repair. Mechanistically, we show that release is governed by two components. First, a conserved glutamate residue, within HMCES' active site, catalyses reversal of the crosslink. Second, affinity to the underlying DNA structure determines whether HMCES re-crosslinks or dissociates. Our study reveals that the protective role of HMCES-DPCs involves their controlled release upon bypass by replication forks, which restricts DPC formation to a necessary minimum.

DNA-protein crosslink repair.

DNA-protein crosslinks (DPCs) are highly toxic DNA adducts, but whether dedicated DPC-repair mechanisms exist was until recently unknown. This has changed with discoveries made in yeast and Xenopus laevis that revealed a protease-based DNA-repair pathway specific for DPCs. Importantly, mutations in the gene encoding the putative human homologue of a yeast DPC protease cause a human premature ageing and cancer predisposition syndrome. Thus, DPC repair is a previously overlooked genome-maintenance mechanism that may be essential for tumour suppression.

Quantification of Intracellular DNA-Protein Cross-Links with N7-Methyl-2'-Deoxyguanosine and Their Contribution to Cytotoxicity.

The major product of DNA-methylating agents, N7-methyl-2'-deoxyguanosine (MdG), is a persistent lesion in vivo, but it is not believed to have a large direct physiological impact. However, MdG reacts with histone proteins to form reversible DNA-protein cross-links (DPCMdG), a family of DNA lesions that can significantly threaten cell survival. In this paper, we developed a tandem mass spectrometry method for quantifying the amounts of MdG and DPCMdG in nuclear DNA by taking advantage of their chemical lability and the concurrent release of N7-methylguanine. Using this method, we determined that DPCMdG is formed in less than 1% yield based upon the levels of MdG in methyl methanesulfonate (MMS)-treated HeLa cells. Despite its low chemical yield, DPCMdG contributes to MMS cytotoxicity. Consequently, cells that lack efficient DPC repair by the DPC protease SPRTN are hypersensitive to MMS. This investigation shows that the downstream chemical and biochemical effects of initially formed DNA damage can have significant biological consequences. With respect to MdG formation, the initial DNA lesion is only the beginning.

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.

A ubiquitin switch controls autocatalytic inactivation of the DNA-protein crosslink repair protease SPRTN.

Repair of covalent DNA-protein crosslinks (DPCs) by the metalloprotease SPRTN prevents genome instability, premature aging and carcinogenesis. SPRTN is specifically activated by DNA structures containing single- and double-stranded features, but degrades the protein components of DPCs promiscuously and independent of amino acid sequence. This lack of specificity is useful to target diverse protein adducts, however, it requires tight control in return, in order to prohibit uncontrolled proteolysis of chromatin proteins. Here, we discover the components and principles of a ubiquitin switch, which negatively regulates SPRTN. We demonstrate that monoubiquitylation is induced in an E3 ligase-independent manner and, in contrast to previous assumptions, does not control chromatin access of the enzyme. Data obtained in cells and in vitro reveal that monoubiquitylation induces inactivation of the enzyme by triggering autocatalytic cleavage in trans while also priming SPRTN for proteasomal degradation in cis. Finally, we show that the deubiquitylating enzyme USP7 antagonizes this negative control of SPRTN in the presence of DPCs.

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.

Genomic barcoding for clonal diversity monitoring and control in cell-based complex antibody production.

Engineered mammalian cells are key for biotechnology by enabling broad applications ranging from in vitro model systems to therapeutic biofactories. Engineered cell lines exist as a population containing sub-lineages of cell clones that exhibit substantial genetic and phenotypic heterogeneity. There is still a limited understanding of the source of this inter-clonal heterogeneity as well as its implications for biotechnological applications. Here, we developed a genomic barcoding strategy for a targeted integration (TI)-based CHO antibody producer cell line development process. This technology provided novel insights about clone diversity during stable cell line selection on pool level, enabled an imaging-independent monoclonality assessment after single cell cloning, and eventually improved hit-picking of antibody producer clones by monitoring of cellular lineages during the cell line development (CLD) process. Specifically, we observed that CHO producer pools generated by TI of two plasmids at a single genomic site displayed a low diversity (< 0.1% RMCE efficiency), which further depends on the expressed molecules, and underwent rapid population skewing towards dominant clones during routine cultivation. Clonal cell lines from one individual TI event demonstrated a significantly lower variance regarding production-relevant and phenotypic parameters as compared to cell lines from distinct TI events. This implies that the observed cellular diversity lies within pre-existing cell-intrinsic factors and that the majority of clonal variation did not develop during the CLD process, especially during single cell cloning. Using cellular barcodes as a proxy for cellular diversity, we improved our CLD screening workflow and enriched diversity of production-relevant parameters substantially. This work, by enabling clonal diversity monitoring and control, paves the way for an economically valuable and data-driven CLD process.

Electro-elution-based purification of covalent DNA-protein cross-links.

Covalent DNA-protein cross-links (DPCs) are pervasive DNA lesions that challenge genome stability and can be induced by metabolic or chemotherapeutic cross-linking agents including reactive aldehydes, topoisomerase poisons and DNMT1 inhibitors. The purification of x-linked proteins (PxP), where DNA-cross-linked proteins are separated from soluble proteins via electro-elution, can be used to identify DPCs. Here we describe a versatile and sensitive strategy for PxP. Mammalian cells are collected following exposure to a DPC-inducing agent, embedded in low-melt agarose plugs and lysed under denaturing conditions. Following lysis, the soluble proteins are extracted from the agarose plug by electro-elution, while genomic DNA and cross-linked proteins are retained in the plug. The cross-linked proteins can then be analyzed by standard analytical techniques such as sodium dodecyl-sulfate-polyacrylamide gel electrophoresis followed by western blotting or fluorescent staining. Alternatively, quantitative mass spectrometry-based proteomics can be used for the unbiased identification of DPCs. The isolation and analysis of DPCs by PxP overcomes the limitations of alternative methods to analyze DPCs that rely on precipitation as the separating principle and can be performed by users trained in molecular or cell biology within 2-3 d. The protocol has been optimized to study DPC induction and repair in mammalian cells but may also be adapted to other sample types including bacteria, yeast and tissue samples.

Transcription-coupled repair of DNA-protein cross-links depends on CSA and CSB.

Covalent DNA-protein cross-links (DPCs) are toxic DNA lesions that block replication and require repair by multiple pathways. Whether transcription blockage contributes to the toxicity of DPCs and how cells respond when RNA polymerases stall at DPCs is unknown. Here we find that DPC formation arrests transcription and induces ubiquitylation and degradation of RNA polymerase II. Using genetic screens and a method for the genome-wide mapping of DNA-protein adducts, DPC sequencing, we discover that Cockayne syndrome (CS) proteins CSB and CSA provide resistance to DPC-inducing agents by promoting DPC repair in actively transcribed genes. Consequently, CSB- or CSA-deficient cells fail to efficiently restart transcription after induction of DPCs. In contrast, nucleotide excision repair factors that act downstream of CSB and CSA at ultraviolet light-induced DNA lesions are dispensable. Our study describes a transcription-coupled DPC repair pathway and suggests that defects in this pathway may contribute to the unique neurological features of CS.

SPRTN patient variants cause global-genome DNA-protein crosslink repair defects.

DNA-protein crosslinks (DPCs) are pervasive DNA lesions that are induced by reactive metabolites and various chemotherapeutic agents. Here, we develop a technique for the Purification of x-linked Proteins (PxP), which allows identification and tracking of diverse DPCs in mammalian cells. Using PxP, we investigate DPC repair in cells genetically-engineered to express variants of the SPRTN protease that cause premature ageing and early-onset liver cancer in Ruijs-Aalfs syndrome patients. We find an unexpected role for SPRTN in global-genome DPC repair, that does not rely on replication-coupled detection of the lesion. Mechanistically, we demonstrate that replication-independent DPC cleavage by SPRTN requires SUMO-targeted ubiquitylation of the protein adduct and occurs in addition to proteasomal DPC degradation. Defective ubiquitin binding of SPRTN patient variants compromises global-genome DPC repair and causes synthetic lethality in combination with a reduction in proteasomal DPC repair capacity.

An arrayed CRISPR screen reveals Myc depletion to increase productivity of difficult-to-express complex antibodies in CHO cells.

Complex therapeutic antibody formats, such as bispecifics (bsAbs) or cytokine fusions, may provide new treatment options in diverse disease areas. However, the manufacturing yield of these complex antibody formats in Chinese Hamster Ovary (CHO) cells is lower than monoclonal antibodies due to challenges in expression levels and potential formation of side products. To overcome these limitations, we performed a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9)-based knockout (KO) arrayed screening of 187 target genes in two CHO clones expressing two different complex antibody formats in a production-mimicking set-up. Our findings revealed that Myc depletion drastically increased product expression (>40%) by enhancing cell-specific productivity. The Myc-depleted cells displayed decreased cell densities together with substantially higher product titers in industrially-relevant bioprocesses using ambr15 and ambr250 bioreactors. Similar effects were observed across multiple different clones, each expressing a distinct complex antibody format. Our findings reinforce the mutually exclusive relationship between growth and production phenotypes and provide a targeted cell engineering approach to impact productivity without impairing product quality. We anticipate that CRISPR/Cas9-based CHO host cell engineering will transform our ability to increase manufacturing yield of high-value complex biotherapeutics.

The yeast E4 ubiquitin ligase Ufd2 interacts with the ubiquitin-like domains of Rad23 and Dsk2 via a novel and distinct ubiquitin-like binding domain.

Proteins containing ubiquitin-like (UBL) and ubiquitin-associated (UBA) domains interact with various binding partners and function as hubs during ubiquitin-mediated protein degradation. A common interaction of the budding yeast UBL-UBA proteins Rad23 and Dsk2 with the E4 ubiquitin ligase Ufd2 has been described in endoplasmic reticulum-associated degradation among other pathways. The UBL domains of Rad23 and Dsk2 play a prominent role in this process by interacting with Ufd2 and different subunits of the 26 S proteasome. Here, we report crystal structures of Ufd2 in complex with the UBL domains of Rad23 and Dsk2. The N-terminal UBL-interacting region of Ufd2 exhibits a unique sequence pattern, which is distinct from any known ubiquitin- or UBL-binding domain identified so far. Residue-specific differences exist in the interactions of these UBL domains with Ufd2, which are coupled to subtle differences in their binding affinities. The molecular details of their differential interactions point to a role for adaptive evolution in shaping these interfaces.