Allosteric activation of the SPRTN protease by ubiquitin maintains genome stability.

The DNA-dependent protease SPRTN maintains genome stability by degrading toxic DNA-protein crosslinks (DPCs). To understand how SPRTN's promiscuous protease activity is confined to cleavage of crosslinked proteins, we reconstitute the repair of DPCs including their modification with SUMO and ubiquitin chains in vitro. We discover that DPC ubiquitylation strongly activates SPRTN independently of SPRTN's known ubiquitin-binding domains.

Reversible association of ubiquitin with PCNA is important for template switching in S. cerevisiae.

Polyubiquitylation of the replication factor PCNA activates the replicative bypass of DNA lesions via an error-free pathway involving template switching. However, the mechanism by which the K63-linked polyubiquitin chains facilitate damage bypass is poorly understood. Intriguingly, stable fusions of linear ubiquitin oligomers to PCNA, designed as mimics of the native K63-linked chains, are not functional, while enzymatic modification of PCNA with linear chains supports template switching in budding yeast.

When DNA Repair Backfires - Trabectedin Induces DNA Breaks in Active Genes.

Many anticancer drugs are ineffective in tumors that have functional DNA repair mechanisms. In contrast, trabectedin, a tetrahydroisoquinoline alkaloid marine natural product, stands out as it is more lethal to cancer cells with active DNA repair, particularly transcription-coupled nucleotide excision repair (TC-NER), making it an intriguing alternative to standard chemotherapeutic agents. To optimize trabectedin's use in precision oncology, it is essential to understand how its toxicity depends on TC-NER.

Post-replicative lesion processing limits DNA damage-induced mutagenesis.

DNA lesions are a threat to genome stability. To cope with them during DNA replication, cells have evolved lesion bypass mechanisms: Translesion Synthesis (TLS), which allows the cell to insert a nucleotide directly opposite the lesion, with the risk of introducing a mutation, and error-free damage avoidance (DA), which uses homologous recombination to retrieve the genetic information from the sister chromatid. In this study, we investigate the timing of lesion bypass in yeast and its implications for the accuracy of the process.

Custom affinity probes reveal DNA-damage-induced, ssDNA-independent chromatin SUMOylation in budding yeast.

The small ubiquitin-related modifier SUMO regulates cellular processes in eukaryotes either by modulating individual protein-protein interactions or with relaxed substrate selectivity by group modification. Here, we report the isolation and characterization of designed ankyrin repeat protein (DARPin)-based affinity probes directed against budding yeast SUMO (Smt3). We validate selected DARPins as compartment-specific inhibitors or neutral detection agents.

TelAP2 links TelAP1 to the telomere complex in Trypanosoma brucei.

The extracellular parasite Trypanosoma brucei evades the immune system of the mammalian host by periodically exchanging its variant surface glycoprotein (VSG) coat. Hereby, only one VSG gene is transcribed from one of 15 subtelomeric so-called bloodstream form expression sites (BES) at any given timepoint, while all other BESs are silenced. VSG gene expression is altered by homologous recombination using a large VSG gene repertoire or by a so-called in situ switch, which activates a previously silent BES.

Analysis of cell cycle stage, replicated DNA, and chromatin-associated proteins using high-throughput flow cytometry.

Flow cytometry is a versatile tool used for cell sorting, DNA content imaging, and determining various cellular characteristics. With the possibility of high-throughput analyses, it combines convenient labelling techniques to serve rapid, quantitative, and qualitative workflows. The ease of sample preparation and the broad range of applications render flow cytometry a preferred approach for many scientific questions.

SNM1A is crucial for efficient repair of complex DNA breaks in human cells.

DNA double-strand breaks (DSBs), such as those produced by radiation and radiomimetics, are amongst the most toxic forms of cellular damage, in part because they involve extensive oxidative modifications at the break termini. Prior to completion of DSB repair, the chemically modified termini must be removed. Various DNA processing enzymes have been implicated in the processing of these dirty ends, but molecular knowledge of this process is limited.

Trabectedin derails transcription-coupled nucleotide excision repair to induce DNA breaks in highly transcribed genes.

Most genotoxic anticancer agents fail in tumors with intact DNA repair. Therefore, trabectedin, anagent more toxic to cells with active DNA repair, specifically transcription-coupled nucleotide excision repair (TC-NER), provides therapeutic opportunities. To unlock the potential of trabectedin and inform its application in precision oncology, an understanding of the mechanism of the drug's TC-NER-dependent toxicity is needed.

K6-linked ubiquitylation marks formaldehyde-induced RNA-protein crosslinks for resolution.

Reactive aldehydes are produced by normal cellular metabolism or after alcohol consumption, and they accumulate in human tissues if aldehyde clearance mechanisms are impaired. Their toxicity has been attributed to the damage they cause to genomic DNA and the subsequent inhibition of transcription and replication. However, whether interference with other cellular processes contributes to aldehyde toxicity has not been investigated.

Chromatin meets the cytoskeleton: the importance of nuclear actin dynamics and associated motors for genome stability.

The actin cytoskeleton is of fundamental importance for numerous cellular processes, including intracellular transport, cell plasticity, and cell migration. However, functions of filamentous actin (F-actin) in the nucleus remain understudied due to the comparatively low abundance of nuclear actin and the resulting experimental limitations to its visualization. Owing to recent technological advances such as super-resolution microscopy and the development of nuclear-specific actin probes, essential roles of the actin cytoskeleton in the context of genome maintenance are now emerging.

Avidity-based biosensors for ubiquitylated PCNA reveal choreography of DNA damage bypass.

In eukaryotes, the posttranslational modifier ubiquitin is used to regulate the amounts, interactions, or activities of proteins in diverse pathways and signaling networks. Its effects are mediated by monoubiquitin or polyubiquitin chains of varying geometries. We describe the design, validation, and application of a series of avidity-based probes against the ubiquitylated forms of the DNA replication clamp, proliferating cell nuclear antigen (PCNA), in budding yeast.

Nuclear myosin VI maintains replication fork stability.

The actin cytoskeleton is of fundamental importance for cellular structure and plasticity. However, abundance and function of filamentous actin in the nucleus are still controversial. Here we show that the actin-based molecular motor myosin VI contributes to the stabilization of stalled or reversed replication forks.

Long non-coding RNA PCAT19 safeguards DNA in quiescent endothelial cells by preventing uncontrolled phosphorylation of RPA2.

In healthy vessels, endothelial cells maintain a stable, differentiated, and growth-arrested phenotype for years. Upon injury, a rapid phenotypic switch facilitates proliferation to restore tissue perfusion. Here we report the identification of the endothelial cell-enriched long non-coding RNA (lncRNA) PCAT19, which contributes to the proliferative switch and acts as a safeguard for the endothelial genome.

Linkage reprogramming by tailor-made E3s reveals polyubiquitin chain requirements in DNA-damage bypass.

A polyubiquitin chain can adopt a variety of shapes, depending on how the ubiquitin monomers are joined. However, the relevance of linkage for the signaling functions of polyubiquitin chains is often poorly understood because of our inability to control or manipulate this parameter in vivo. Here, we present a strategy for reprogramming polyubiquitin chain linkage by means of tailor-made, linkage- and substrate-selective ubiquitin ligases.

Fast friends - Ubiquitin-like modifiers as engineered fusion partners.

Ubiquitin and its relatives are major players in many biological pathways, and a variety of experimental tools based on biological chemistry or protein engineering is available for their manipulation. One popular approach is the use of linear fusions between the modifier and a protein of interest. Such artificial constructs can facilitate the understanding of the role of ubiquitin in biological processes and can be exploited to control protein stability, interactions and degradation.

Physical interactions between MCM and Rad51 facilitate replication fork lesion bypass and ssDNA gap filling by non-recombinogenic functions.

The minichromosome maintenance (MCM) helicase physically interacts with the recombination proteins Rad51 and Rad52 from yeast to human cells. We show, in Saccharomyces cerevisiae, that these interactions occur within a nuclease-insoluble scaffold enriched in replication/repair factors. Rad51 accumulates in a MCM- and DNA-binding-independent manner and interacts with MCM helicases located outside of the replication origins and forks.

Daughter-strand gaps in DNA replication - substrates of lesion processing and initiators of distress signalling.

Dealing with DNA lesions during genome replication is particularly challenging because damaged replication templates interfere with the progression of the replicative DNA polymerases and thereby endanger the stability of the replisome. A variety of mechanisms for the recovery of replication forks exist, but both bacteria and eukaryotic cells also have the option of continuing replication downstream of the lesion, leaving behind a daughter-strand gap in the newly synthesized DNA. In this review, we address the significance of these single-stranded DNA structures as sites of DNA damage sensing and processing at a distance from ongoing genome replication.

The double-stranded DNA-binding proteins TEBP-1 and TEBP-2 form a telomeric complex with POT-1.

Telomeres are bound by dedicated proteins, which protect them from DNA damage and regulate telomere length homeostasis. In the nematode Caenorhabditis elegans, a comprehensive understanding of the proteins interacting with the telomere sequence is lacking. Here, we harnessed a quantitative proteomics approach to identify TEBP-1 and TEBP-2, two paralogs expressed in the germline and embryogenesis that associate to telomeres in vitro and in vivo.

Global non-covalent SUMO interaction networks reveal SUMO-dependent stabilization of the non-homologous end joining complex.

In contrast to our extensive knowledge on covalent small ubiquitin-like modifier (SUMO) target proteins, we are limited in our understanding of non-covalent SUMO-binding proteins. We identify interactors of different SUMO isoforms-monomeric SUMO1, monomeric SUMO2, or linear trimeric SUMO2 chains-using a mass spectrometry-based proteomics approach. We identify 379 proteins that bind to different SUMO isoforms, mainly in a preferential manner.

Non-recombinogenic roles for Rad52 in translesion synthesis during DNA damage tolerance.

DNA damage tolerance relies on homologous recombination (HR) and translesion synthesis (TLS) mechanisms to fill in the ssDNA gaps generated during passing of the replication fork over DNA lesions in the template. Whereas TLS requires specialized polymerases able to incorporate a dNTP opposite the lesion and is error-prone, HR uses the sister chromatid and is mostly error-free. We report that the HR protein Rad52-but not Rad51 and Rad57-acts in concert with the TLS machinery (Rad6/Rad18-mediated PCNA ubiquitylation and polymerases Rev1/Pol ζ) to repair MMS and UV light-induced ssDNA gaps through a non-recombinogenic mechanism, as inferred from the different phenotypes displayed in the absence of Rad52 and Rad54 (essential for MMS- and UV-induced HR); accordingly, Rad52 is required for efficient DNA damage-induced mutagenesis.

Preparation and Analysis of GLOE-Seq Libraries for Genome-Wide Mapping of DNA Replication Patterns, Single-Strand Breaks, and Lesions.

GLOE-Seq is a next-generation sequencing method for the genome-wide mapping of 3'-OH termini, either resulting from single- or double-strand breaks or introduced by enzymatic conversion of lesions or modified nucleotides. This protocol provides instructions for isolation of genomic DNA from budding yeast or mammalian cells, preparation of libraries for sequencing, and data analysis by the associated computational pipeline, GLOE-Pipe. It is optimized for the Illumina next-generation sequencing platform and can be adapted to intact genomic DNA of any origin.