Protein-oligonucleotide conjugates as model substrates for DNA-protein crosslink repair proteases.
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ABSTRACT:
SUBMITTER: Reinking HK
PROVIDER: S-EPMC8219891 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Proteins covalently attached to DNA, also known as DNA-protein crosslinks (DPCs), are common and bulky DNA lesions that interfere with DNA replication, repair, transcription and recombination. Research in the past several years indicates that cells possess dedicated enzymes, known as DPC proteases, which digest the protein component of a DPC. Interestingly, DPC proteases also play a role in proteolysis beside DPC repair, such as in degrading excess histones during DNA replication or controlling DNA replication checkpoints. Here, we discuss the importance of DPC proteases in DNA replication, genome stability and their direct link to human diseases and cancer therapy.
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Project description:DNA-protein crosslinks (DPCs) are caused by environmental, endogenous, and chemotherapeutic agents and pose a severe threat to genome stability. We use Xenopus egg extracts to recapitulate DPC repair in vitro and show that this process is coupled to DNA replication. A DPC on the leading strand template arrests the replisome by stalling the CMG helicase. The DPC is then degraded on DNA, yielding a peptide-DNA adduct that is bypassed by CMG. The leading strand subsequently resumes synthesis, stalls again at the adduct, and then progresses past the adduct using DNA polymerase ?. A DPC on the lagging strand template only transiently stalls the replisome, but it too is degraded, allowing Okazaki fragment bypass. Our experiments describe a versatile, proteolysis-based mechanism of S phase DPC repair that avoids replication fork collapse.
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Project description: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.
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Project description:DNA-protein crosslinks (DPCs) are toxic lesions that inhibit DNA related processes. Post-translational modifications (PTMs), including SUMOylation and ubiquitylation, play a central role in DPC resolution, but whether other PTMs are also involved remains elusive. Here, we identify a DPC repair pathway orchestrated by poly-ADP-ribosylation (PARylation). Using Xenopus egg extracts, we show that DPCs on single-stranded DNA gaps can be targeted for degradation via a replication-independent mechanism. During this process, DPCs are initially PARylated by PARP1 and subsequently ubiquitylated and degraded by the proteasome. Notably, PARP1-mediated DPC resolution is required for resolving topoisomerase 1-DNA cleavage complexes (TOP1ccs) induced by camptothecin. Using the Flp-nick system, we further reveal that in the absence of PARP1 activity, the TOP1cc-like lesion persists and induces replisome disassembly when encountered by a DNA replication fork. In summary, our work uncovers a PARP1-mediated DPC repair pathway that may underlie the synergistic toxicity between TOP1 poisons and PARP inhibitors.
2024-08-06 | PXD043107 | Pride
Project description: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.
| S-EPMC5128726 | biostudies-other
Project description:DNA interstrand crosslinks (ICLs) are toxic DNA lesions whose repair occurs in the S phase of metazoans via an unknown mechanism. Here, we describe a cell-free system based on Xenopus egg extracts that supports ICL repair. During DNA replication of a plasmid containing a site-specific ICL, two replication forks converge on the crosslink. Subsequent lesion bypass involves advance of a nascent leading strand to within one nucleotide of the ICL, followed by incisions, translesion DNA synthesis, and extension of the nascent strand beyond the lesion. Immunodepletion experiments suggest that extension requires DNA polymerase zeta. Ultimately, a significant portion of the input DNA is fully repaired, but not if DNA replication is blocked. Our experiments establish a mechanism for ICL repair that reveals how this process is coupled to DNA replication.
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Project description:DNA-protein crosslinks (DPCs) obstruct essential DNA transactions, posing a serious threat to genome stability and functionality. DPCs are proteolytically processed in a ubiquitin- and DNA replication-dependent manner by SPRTN and the proteasome but can also be resolved via targeted SUMOylation. However, the mechanistic basis of SUMO-mediated DPC resolution and its interplay with replication-coupled DPC repair remain unclear. Here, we show that the SUMO-targeted ubiquitin ligase RNF4 defines a major pathway for ubiquitylation and proteasomal clearance of SUMOylated DPCs in the absence of DNA replication. Importantly, SUMO modifications of DPCs neither stimulate nor inhibit their rapid DNA replication-coupled proteolysis. Instead, DPC SUMOylation provides a critical salvage mechanism to remove DPCs formed after DNA replication, as DPCs on duplex DNA do not activate interphase DNA damage checkpoints. Consequently, in the absence of the SUMO-RNF4 pathway cells are able to enter mitosis with a high load of unresolved DPCs, leading to defective chromosome segregation and cell death. Collectively, these findings provide mechanistic insights into SUMO-driven pathways underlying replication-independent DPC resolution and highlight their critical importance in maintaining chromosome stability and cellular fitness.
| S-EPMC8441304 | biostudies-literature
Project description:The repair mechanisms acting on DNA interstrand crosslinks (ICLs) in eukaryotes are poorly understood. Here, we provide evidence for a pathway of ICL processing that uses components from both nucleotide excision repair (NER) and translesion synthesis (TLS) and predominates during the G1 phase of the yeast cell cycle. Our results suggest that repair is initiated by the NER apparatus and is followed by a thwarted attempt at gap-filling by the replicative Polymerase delta, which likely stalls at the site of the remaining crosslinked oligonucleotide. This in turn leads to ubiquitination of PCNA and recruitment of the damage-tolerant Polymerase zeta that can perform TLS. The ICL repair factor Pso2 acts downstream of the incision step and is not required for Polymerase zeta activation. We show that this combination of NER and TLS is the only pathway of ICL repair available to the cell in G1 phase and is essential for viability in the presence of DNA crosslinks.
| S-EPMC1422152 | biostudies-literature
Project description:DNA-protein crosslinks (DPCs) are a specific type of DNA lesion consisting of a protein covalently and irreversibly bound to DNA, which arise after exposure to physical and chemical crosslinking agents. DPCs can be bulky and thereby pose a barrier to DNA replication and transcription. The persistence of DPCs during S phase causes DNA replication stress and genome instability. The toxicity of DPCs is exploited in cancer therapy: many common chemotherapeutics kill cancer cells by inducing DPC formation. Recent work from several laboratories discovered a specialized repair pathway for DPCs, namely DPC proteolysis (DPCP) repair. DPCP repair is carried out by replication-coupled DNA-dependent metalloproteases: Wss1 in yeast and SPRTN in metazoans. Mutations in SPRTN cause premature ageing and liver cancer in humans and mice; thus, defective DPC repair has great clinical ramifications. In the present review, we will revise the current knowledge on the mechanisms of DPCP repair and on the regulation of DPC protease activity, while highlighting the most significant unresolved questions in the field. Finally, we will discuss the impact of faulty DPC repair on disease and cancer therapy.
| S-EPMC6219452 | biostudies-literature
Project description:DNA-protein crosslinks (DPCs) are a specific type of DNA lesion in which proteins are covalently attached to DNA. Unrepaired DPCs lead to genomic instability, cancer, neurodegeneration, and accelerated aging. DPC proteolysis was recently identified as a specialized pathway for DPC repair. The DNA-dependent protease SPRTN and the 26S proteasome emerged as two independent proteolytic systems. DPCs are also repaired by homologous recombination (HR), a canonical DNA repair pathway. While studying the cellular response to DPC formation, we identify ubiquitylation and SUMOylation as two major signaling events in DNA replication-coupled DPC repair. DPC ubiquitylation recruits SPRTN to repair sites, promoting DPC removal. DPC SUMOylation prevents DNA double-strand break formation, HR activation, and potentially deleterious genomic rearrangements. In this way, SUMOylation channels DPC repair toward SPRTN proteolysis, which is a safer pathway choice for DPC repair and prevention of genomic instability.
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