Mammalian cells frequently enter mitosis before DNA replication has finished, necessitating the rapid processing of unreplicated loci to facilitate chromosome segregation. The TRAIP ubiquitin ligase induces replisome disassembly during mitosis, triggering the cleavage of DNA replication forks. Until now, the mechanisms that regulate TRAIP and process cleaved DNA replication forks were unclear. In this study, we show that the transcription termination factor 2 (TTF2) adenosine triphosphatase is a new type of phosphoreceptor that binds a conserved phosphorylation site on TRAIP during mitosis. TTF2 couples phosphorylated TRAIP to DNA polymerase ε (Pol ε) in the replisome, leading TRAIP to ubiquitylate the CDC45-MCM-GINS (CMG) helicase. This triggers mitotic replisome disassembly and a repair pathway that produces sister-chromatid exchanges, supporting a model for how fork cleavage promotes the segregation of underreplicated loci in mammalian cells.

Mammalian cells frequently enter mitosis before DNA replication has finished, necessitating the rapid processing of replication forks to facilitate chromosome segregation. The TRAIP ubiquitin ligase induces mitotic replisome disassembly, fork cleavage, and repair via Mitotic DNA Synthesis (MiDAS). Until now, it was unclear how TRAIP is regulated in mitotic cells. Here we show that TRAIP phosphorylation mediates a complex with the TTF2 ATPase and DNA Polymerase epsilon (Pol epsilon). Whereas TTF2 ATPase activity removes RNA polymerase II from mitotic chromosomes, replisome disassembly involves an unanticipated mechanism. The TTF2 amino terminus couples TRAIP to Pol epsilon, via tandem Zinc fingers that recognise phosphorylated TRAIP, and a motif that binds to POLE2. Thereby, TTF2 and Pol epsilon cause TRAIP to ubiquitylate the CDC45-MCM-GINS (CMG) helicase, triggering replisome disassembly and MiDAS. These data identify TTF2 as a multifunctional regulator of chromatin transactions during mitosis. ### Competing Interest Statement The authors have declared no competing interest.

The list of antibodies used in this study.

DONSON is one of 13 genes mutated in a form of primordial microcephalic dwarfism known as Meier-Gorlin syndrome. The other 12 encode components of the CDC45-MCM-GINS helicase, around which the eukaryotic replisome forms, or are factors required for helicase assembly during DNA replication initiation. A role for DONSON in CDC45-MCM-GINS assembly was unanticipated, since DNA replication initiation can be reconstituted in vitro with purified proteins from budding yeast, which lacks DONSON. Using mouse embryonic stem cells as a model for the mammalian helicase, we show that DONSON binds directly but transiently to CDC45-MCM-GINS during S-phase and is essential for chromosome duplication. Rapid depletion of DONSON leads to the disappearance of the CDC45-MCM-GINS helicase from S-phase cells and our data indicate that DONSON is dispensable for loading of the MCM2-7 helicase core onto chromatin during G1-phase, but instead is essential for CDC45-MCM-GINS assembly during S-phase. These data identify DONSON as a missing link in our understanding of mammalian chromosome duplication and provide a molecular explanation for why mutations in human DONSON are associated with Meier-Gorlin syndrome.

The p97/Cdc48 ATPase and its ubiquitin receptors Ufd1-Npl4 are essential to unfold ubiquitylated proteins in many areas of eukaryotic cell biology. In yeast, Cdc48-Ufd1-Npl4 is controlled by a quality control mechanism, whereby substrates must be conjugated to at least five ubiquitins. Here, we show that mammalian p97-UFD1-NPL4 is governed by a complex interplay between additional p97 cofactors and the number of conjugated ubiquitins. Using reconstituted assays for the disassembly of ubiquitylated CMG (Cdc45-MCM-GINS) helicase by human p97-UFD1-NPL4, we show that the unfoldase has a high ubiquitin threshold for substrate unfolding, which can be reduced by the UBX proteins UBXN7, FAF1, or FAF2. Our data indicate that the UBX proteins function by binding to p97-UFD1-NPL4 and stabilising productive interactions between UFD1-NPL4 and K48-linked chains of at least five ubiquitins. Stimulation by UBXN7 is dependent upon known ubiquitin-binding motifs, whereas FAF1 and FAF2 use a previously uncharacterised coiled-coil domain to reduce the ubiquitin threshold of p97-UFD1-NPL4. We show that deleting the Ubnx7 and Faf1 genes impairs CMG disassembly during S-phase and mitosis and sensitises cells to reduced ubiquitin ligase activity. These findings indicate that multiple UBX proteins are important for the efficient unfolding of ubiquitylated proteins by p97-UFD1-NPL4 in mammalian cells.

SARS-CoV-2 is responsible for COVID-19, a human disease that has caused over 2 million deaths, stretched health systems to near-breaking point and endangered economies of countries and families around the world. Antiviral treatments to combat COVID-19 are currently lacking. Remdesivir, the only antiviral drug approved for the treatment of COVID-19, can affect disease severity, but better treatments are needed. SARS-CoV-2 encodes 16 non-structural proteins (nsp) that possess different enzymatic activities with important roles in viral genome replication, transcription and host immune evasion. One key aspect of host immune evasion is performed by the uridine-directed endoribonuclease activity of nsp15. Here we describe the expression and purification of nsp15 recombinant protein. We have developed biochemical assays to follow its activity, and we have found evidence for allosteric behaviour. We screened a custom chemical library of over 5000 compounds to identify nsp15 endoribonuclease inhibitors, and we identified and validated NSC95397 as an inhibitor of nsp15 endoribonuclease in vitro. Although NSC95397 did not inhibit SARS-CoV-2 growth in VERO E6 cells, further studies will be required to determine the effect of nsp15 inhibition on host immune evasion.

The COVID-19 pandemic has presented itself as one of the most critical public health challenges of the century, with SARS-CoV-2 being the third member of the Coronaviridae family to cause a fatal disease in humans. There is currently only one antiviral compound, remdesivir, that can be used for the treatment of COVID-19. To identify additional potential therapeutics, we investigated the enzymatic proteins encoded in the SARS-CoV-2 genome. In this study, we focussed on the viral RNA cap methyltransferases, which play key roles in enabling viral protein translation and facilitating viral escape from the immune system. We expressed and purified both the guanine-N7 methyltransferase nsp14, and the nsp16 2′-O-methyltransferase with its activating cofactor, nsp10. We performed an in vitro high-throughput screen for inhibitors of nsp14 using a custom compound library of over 5000 pharmaceutical compounds that have previously been characterised in either clinical or basic research. We identified four compounds as potential inhibitors of nsp14, all of which also showed antiviral capacity in a cell-based model of SARS-CoV-2 infection. Three of the four compounds also exhibited synergistic effects on viral replication with remdesivir.

SARS-CoV-2 is a coronavirus that emerged in 2019 and rapidly spread across the world causing a deadly pandemic with tremendous social and economic costs. Healthcare systems worldwide are under great pressure, and there is an urgent need for effective antiviral treatments. The only currently approved antiviral treatment for COVID-19 is remdesivir, an inhibitor of viral genome replication. SARS-CoV-2 proliferation relies on the enzymatic activities of the non-structural proteins (nsp), which makes them interesting targets for the development of new antiviral treatments. With the aim to identify novel SARS-CoV-2 antivirals, we have purified the exoribonuclease/methyltransferase (nsp14) and its cofactor (nsp10) and developed biochemical assays compatible with high-throughput approaches to screen for exoribonuclease inhibitors. We have screened a library of over 5000 commercial compounds and identified patulin and aurintricarboxylic acid (ATA) as inhibitors of nsp14 exoribonuclease in vitro. We found that patulin and ATA inhibit replication of SARS-CoV-2 in a VERO E6 cell-culture model. These two new antiviral compounds will be valuable tools for further coronavirus research as well as potentially contributing to new therapeutic opportunities for COVID-19.

The eukaryotic replisome is disassembled in each cell cycle, dependent upon ubiquitylation of the CMG helicase. Studies of Saccharomyces cerevisiae, Caenorhabditis elegans and Xenopus laevis have revealed surprising evolutionary diversity in the ubiquitin ligases that control CMG ubiquitylation, but regulated disassembly of the mammalian replisome has yet to be explored. Here, we describe a model system for studying the ubiquitylation and chromatin extraction of the mammalian CMG replisome, based on mouse embryonic stem cells. We show that the ubiquitin ligase CUL2LRR1 is required for ubiquitylation of the CMG-MCM7 subunit during S-phase, leading to disassembly by the p97 ATPase. Moreover, a second pathway of CMG disassembly is activated during mitosis, dependent upon the TRAIP ubiquitin ligase that is mutated in primordial dwarfism and mis-regulated in various cancers. These findings indicate that replisome disassembly in diverse metazoa is regulated by a conserved pair of ubiquitin ligases, distinct from those present in other eukaryotes.

The eukaryotic replisome is rapidly disassembled during DNA replication termination. In metazoa, the cullin‐RING ubiquitin ligase CUL‐2LRR‐1 drives ubiquitylation of the CMG helicase, leading to replisome disassembly by the p97/CDC‐48 “unfoldase”. Here, we combine in vitro reconstitution with in vivo studies in Caenorhabditis elegans embryos, to show that the replisome‐associated TIMELESS‐TIPIN complex is required for CUL‐2LRR‐1 recruitment and efficient CMG helicase ubiquitylation. Aided by TIMELESS‐TIPIN, CUL‐2LRR‐1 directs a suite of ubiquitylation enzymes to ubiquitylate the MCM‐7 subunit of CMG. Subsequently, the UBXN‐3 adaptor protein directly stimulates the disassembly of ubiquitylated CMG by CDC‐48_UFD‐1_NPL‐4. We show that UBXN‐3 is important in vivo for replisome disassembly in the absence of TIMELESS‐TIPIN. Correspondingly, co‐depletion of UBXN‐3 and TIMELESS causes profound synthetic lethality. Since the human orthologue of UBXN‐3, FAF1, is a candidate tumour suppressor, these findings suggest that manipulation of CMG disassembly might be applicable to future strategies for treating human cancer. Different factors contribute to ubiquitylation‐mediated DNA replication termination in different eukaryotic systems. Here, replisome disassembly in Caenorhabditis elegans is found to be stimulated by the replisome‐associated TIMELESS‐TIPIN complex and the p97/CDC‐48 unfoldase adaptor UBXN‐3. CUL2/LRR1‐dependent CMG helicase ubiquitination is induced by replisome‐associated TIM‐1/TIPN‐1 and further processed by the p97/CDC‐48 unfoldase stimulated by the UFD‐1, NPL‐4 and UBXN‐3 adaptor proteins.

The coronavirus 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), spread around the world with unprecedented health and socio-economic effects for the global population. While different vaccines are now being made available, very few antiviral drugs have been approved. The main viral protease (nsp5) of SARS-CoV-2 provides an excellent target for antivirals, due to its essential and conserved function in the viral replication cycle. We have expressed, purified and developed assays for nsp5 protease activity. We screened the nsp5 protease against a custom chemical library of over 5,000 characterised pharmaceuticals. We identified calpain inhibitor I and three different peptidyl fluoromethylketones (FMK) as inhibitors of nsp5 activity in vitro, with IC50 values in the low micromolar range. By altering the sequence of our peptidomimetic FMK inhibitors to better mimic the substrate sequence of nsp5, we generated an inhibitor with a subnanomolar IC50. Calpain inhibitor I inhibited viral infection in monkey-derived Vero E6 cells, with an EC50 in the low micromolar range. The most potent and commercially available peptidyl-FMK compound inhibited viral growth in Vero E6 cells to some extent, while our custom peptidyl FMK inhibitor offered a marked antiviral improvement.

Abstract DNA replication stress (DRS) is a predominant cause of genome instability, a driver of tumorigenesis and malignant progression. Nucleoside analogue-type chemotherapeutic drugs introduce DNA damage and exacerbate DRS in tumor cells. However, the mechanisms underlying the antitumor effect of these drugs are not fully understood. Here, we show that the fluorinated thymidine analogue trifluridine (FTD), an active component of the chemotherapeutic drug trifluridine/tipiracil, delayed DNA synthesis by human replicative DNA polymerases by acting both as an inefficient deoxyribonucleotide triphosphate source (FTD triphosphate) and as an obstacle base (trifluorothymine) in the template DNA strand, which caused DRS. In cells, FTD decreased the thymidine triphosphate level in the dNTP pool and increased the FTD triphosphate level, resulting in the activation of DRS-induced cellular responses during S-phase. In addition, replication protein A–coated single-stranded DNA associated with FancD2 and accumulated after tumor cells completed S-phase. Finally, FTD activated the p53–p21 pathway and suppressed tumor cell growth by inducing cellular senescence via mitosis skipping. In contrast, tumor cells that lost wild-type p53 underwent apoptotic cell death via aberrant late mitosis with severely impaired separation of sister chromatids. These results demonstrate that DRS induced by a nucleoside analogue–type chemotherapeutic drug suppresses tumor growth irrespective of p53 status by directing tumor cell fate toward cellular senescence or apoptotic cell death according to p53 status. Implications: Chemotherapeutic drugs that increase DRS during S-phase but allow tumor cells to complete S-phase may have significant antitumor activity even when functional p53 is lost.

The eukaryotic replisome assembles around the CMG helicase, which stably associates with DNA replication forks throughout elongation. When replication terminates, CMG is ubiquitylated on its Mcm7 subunit and disassembled by the Cdc48/p97 ATPase. Until now, the regulation that restricts CMG ubiquitylation to termination was unknown, as was the mechanism of disassembly. By reconstituting these processes with purified budding yeast proteins, we show that ubiquitylation is tightly repressed throughout elongation by the Y-shaped DNA structure of replication forks. Termination removes the repressive DNA structure, whereupon long K48-linked ubiquitin chains are conjugated to CMG-Mcm7, dependent on multiple replisome components that bind to the ubiquitin ligase SCFDia2. This mechanism pushes CMG beyond a '5-ubiquitin threshold' that is inherent to Cdc48, which specifically unfolds ubiquitylated Mcm7 and thereby disassembles CMG. These findings explain the exquisite regulation of CMG disassembly and provide a general model for the disassembly of ubiquitylated protein complexes by Cdc48.

Abstract The alternative proliferating-cell nuclear antigen (PCNA)-loader CTF18-RFC forms a stable complex with DNA polymerase ε (Polε). We observed that, under near-physiological conditions, CTF18-RFC alone loaded PCNA inefficiently, but loaded it efficiently when complexed with Polε. During efficient PCNA loading, CTF18-RFC and Polε assembled at a 3΄ primer–template junction cooperatively, and directed PCNA to the loading site. Site-specific photo-crosslinking of directly interacting proteins at the primer–template junction showed similar cooperative binding, in which the catalytic N-terminal portion of Polε acted as the major docking protein. In the PCNA-loading intermediate with ATPγS, binding of CTF18 to the DNA structures increased, suggesting transient access of CTF18-RFC to the primer terminus. Polε placed in DNA synthesis mode using a substrate DNA with a deoxidised 3΄ primer end did not stimulate PCNA loading, suggesting that DNA synthesis and PCNA loading are mutually exclusive at the 3΄ primer–template junction. Furthermore, PCNA and CTF18-RFC–Polε complex engaged in stable trimeric assembly on the template DNA and synthesised DNA efficiently. Thus, CTF18-RFC appears to be involved in leading-strand DNA synthesis through its interaction with Polε, and can load PCNA onto DNA when Polε is not in DNA synthesis mode to restore DNA synthesis.

Proliferating cell nuclear antigen (PCNA) is a multifunctional protein present in the nuclei of eukaryotic cells that plays an important role as a component of the DNA replication machinery, as well as DNA repair systems. PCNA was recently proposed as a potential non-oncogenic target for anti-cancer therapy. In this study, using the Systematic Evolution of Ligands by EXponential enrichment (SELEX) method, we developed a short DNA aptamer that binds human PCNA. In the presence of PCNA, the anti-PCNA aptamer inhibited the activity of human DNA polymerase δ and ϵ at nM concentrations. Moreover, PCNA protected the anti-PCNA aptamer against the exonucleolytic activity of these DNA polymerases. Investigation of the mechanism of anti-PCNA aptamer-dependent inhibition of DNA replication revealed that the aptamer did not block formation, but was a component of PCNA/DNA polymerase δ or ϵ complexes. Additionally, the anti-PCNA aptamer competed with the primer-template DNA for binding to the PCNA/DNA polymerase δ or ϵ complex. Based on the observations, a model of anti-PCNA aptamer/PCNA complex-dependent inhibition of DNA replication was proposed.

Chemotherapeutic nucleoside analogs, such as Ara-C, 5-Fluorouracil (5-FU) and Trifluridine (FTD), are frequently incorporated into DNA by the replicative DNA polymerases. However, it remains unclear how this incorporation kills cycling cells. There are two possibilities: Nucleoside analog triphosphates inhibit the replicative DNA polymerases, and/or nucleotide analogs mis-incorporated into genomic DNA interfere with the next round of DNA synthesis as replicative DNA polymerases recognize them as template DNA lesions, arresting synthesis. To address the first possibility, we selectively disrupted the proofreading exonuclease activity of DNA polymerase ε (Polε), the leading-strand replicative polymerase in avian DT40 and human TK6 cell lines. To address the second, we disrupted RAD18, a gene involved in translesion DNA synthesis, a mechanism that relieves stalled replication. Strikingly, POLE1exo-/- cells, but not RAD18-/- cells, were hypersensitive to Ara-C, while RAD18-/- cells were hypersensitive to FTD. gH2AX focus formation following a pulse of Ara-C was immediate and did not progress into the next round of replication, while gH2AX focus formation following a pulse of 5-FU and FTD was delayed to the next round of replication. Biochemical studies indicate that human proofreading-deficient Polε-exo- holoenzyme incorporates Ara-CTP, but subsequently extend from this base several times less efficiently than from intact nucleotides. Together our results suggest that Ara-C acts by blocking extension of the nascent DNA strand and is counteracted by the proofreading activity of Polε, while 5-FU and FTD are efficiently incorporated but act as replication fork blocks in the subsequent S phase, which is counteracted by translesion synthesis.