Chk1 loss creates replication barriers that compromise cell survival independently of excess origin firing.

The effectiveness of checkpoint kinase 1 (Chk1) inhibitors at killing cancer cells is considered to be fully dependent on their effect on DNA replication initiation. Chk1 inhibition boosts origin firing, presumably limiting the availability of nucleotides and in turn provoking the slowdown and subsequent collapse of forks, thus decreasing cell viability. Here we show that slow fork progression in Chk1-inhibited cells is not an indirect effect of excess new origin firing. Instead, fork slowdown results from the accumulation of replication barriers, whose bypass is impeded by CDK-dependent phosphorylation of the specialized DNA polymerase eta (Polη). Also in contrast to the linear model, the accumulation of DNA damage in Chk1-deficient cells depends on origin density but is largely independent of fork speed. Notwithstanding this, origin dysregulation contributes only mildly to the poor proliferation rates of Chk1-depleted cells. Moreover, elimination of replication barriers by downregulation of helicase components, but not their bypass by Polη, improves cell survival. Our results thus shed light on the molecular basis of the sensitivity of tumors to Chk1 inhibition.

DROSHA is recruited to DNA damage sites by the MRN complex to promote non-homologous end joining.

The DNA damage response (DDR) is the signaling cascade that recognizes DNA double-strand breaks (DSBs) and promotes their resolution via the DNA repair pathways of non-homologous end joining (NHEJ) or homologous recombination (HR). We and others have shown that DDR activation requires DROSHA; however, whether DROSHA exerts its functions by associating with damage sites, what controls its recruitment, and how DROSHA influences DNA repair remains poorly understood. Here, we show that DROSHA associates with DSBs independently of transcription. Neither H2AX, nor ATM or DNA-PK kinase activities are required for recruitment of DROSHA to break sites. Rather, DROSHA interacts with RAD50, and inhibition of the MRN complex by mirin treatment abolishes this interaction. MRN complex inactivation by RAD50 knockdown or mirin treatment prevents DROSHA recruitment to DSBs and, as a consequence, also prevents 53BP1 (also known as TP53BP1) recruitment. During DNA repair, DROSHA inactivation reduces NHEJ and boosts HR frequency. Indeed, DROSHA knockdown also increases the association of downstream HR factors such as RAD51 to DNA ends. Overall, our results demonstrate that DROSHA is recruited at DSBs by the MRN complex and directs DNA repair towards NHEJ.

Elongating RNA polymerase II and RNA:DNA hybrids hinder fork progression and gene expression at sites of head-on replication-transcription collisions.

Uncoordinated clashes between replication forks and transcription cause replication stress and genome instability, which are hallmarks of cancer and neurodegeneration. Here, we investigate the outcomes of head-on replication-transcription collisions, using as a model system budding yeast mutants for the helicase Sen1, the ortholog of human Senataxin. We found that RNA Polymerase II accumulates together with RNA:DNA hybrids at sites of head-on collisions. The replication fork and RNA Polymerase II are both arrested during the clash, leading to DNA damage and, in the long run, the inhibition of gene expression. The inactivation of RNA Polymerase II elongation factors, such as the HMG-like protein Spt2 and the DISF and PAF complexes, but not alterations in chromatin structure, allows replication fork progression through transcribed regions. Attenuation of RNA Polymerase II elongation rescues RNA:DNA hybrid accumulation and DNA damage sensitivity caused by the absence of Sen1, but not of RNase H proteins, suggesting that such enzymes counteract toxic RNA:DNA hybrids at different stages of the cell cycle with Sen1 mainly acting in replication. We suggest that the main obstacle to replication fork progression is the elongating RNA Polymerase II engaged in an R-loop, rather than RNA:DNA hybrids per se or hybrid-associated chromatin modifications.

Novel alternative ribonucleotide excision repair pathways in human cells by DDX3X and specialized DNA polymerases.

Removal of ribonucleotides (rNMPs) incorporated into the genome by the ribonucleotide excision repair (RER) is essential to avoid genetic instability. In eukaryotes, the RNaseH2 is the only known enzyme able to incise 5' of the rNMP, starting the RER process, which is subsequently carried out by replicative DNA polymerases (Pols) δ or ϵ, together with Flap endonuclease 1 (Fen-1) and DNA ligase 1. Here, we show that the DEAD-box RNA helicase DDX3X has RNaseH2-like activity and can support fully reconstituted in vitro RER reactions, not only with Pol δ but also with the repair Pols ß and λ. Silencing of DDX3X causes accumulation of rNMPs in the cellular genome. These results support the existence of alternative RER pathways conferring high flexibility to human cells in responding to the threat posed by rNMPs incorporation.

UBR5 interacts with the replication fork and protects DNA replication from DNA polymerase η toxicity.

Accurate DNA replication is critical for the maintenance of genome integrity and cellular survival. Cancer-associated alterations often involve key players of DNA replication and of the DNA damage-signalling cascade. Post-translational modifications play a fundamental role in coordinating replication and repair and central among them is ubiquitylation. We show that the E3 ligase UBR5 interacts with components of the replication fork, including the translesion synthesis (TLS) polymerase polη. Depletion of UBR5 leads to replication problems, such as slower S-phase progression, resulting in the accumulation of single stranded DNA. The effect of UBR5 knockdown is related to a mis-regulation in the pathway that controls the ubiquitylation of histone H2A (UbiH2A) and blocking this modification is sufficient to rescue the cells from replication problems. We show that the presence of polη is the main cause of replication defects and cell death when UBR5 is silenced. Finally, we unveil a novel interaction between polη and H2A suggesting that UbiH2A could be involved in polη recruitment to the chromatin and the regulation of TLS.

A Role for Human DNA Polymerase λ in Alternative Lengthening of Telomeres.

Telomerase negative cancer cell types use the Alternative Lengthening of Telomeres (ALT) pathway to elongate telomeres ends. Here, we show that silencing human DNA polymerase (Pol λ) in ALT cells represses ALT activity and induces telomeric stress. In addition, replication stress in the absence of Pol λ, strongly affects the survival of ALT cells. In vitro, Pol λ can promote annealing of even a single G-rich telomeric repeat to its complementary strand and use it to prime DNA synthesis. The noncoding telomeric repeat containing RNA TERRA and replication protein A negatively regulate this activity, while the Protection of Telomeres protein 1 (POT1)/TPP1 heterodimer stimulates Pol λ. Pol λ associates with telomeres and colocalizes with TPP1 in cells. In summary, our data suggest a role of Pol λ in the maintenance of telomeres by the ALT mechanism.

From R-Loops to G-Quadruplexes: Emerging New Threats for the Replication Fork.

Replicating the entire genome is one of the most complex tasks for all organisms. Research carried out in the last few years has provided us with a clearer picture on how cells preserve genomic information from the numerous insults that may endanger its stability. Different DNA repair pathways, coping with exogenous or endogenous threat, have been dissected at the molecular level. More recently, there has been an increasing interest towards intrinsic obstacles to genome replication, paving the way to a novel view on genomic stability. Indeed, in some cases, the movement of the replication fork can be hindered by the presence of stable DNA: RNA hybrids (R-loops), the folding of G-rich sequences into G-quadruplex structures (G4s) or repetitive elements present at Common Fragile Sites (CFS). Although differing in their nature and in the way they affect the replication fork, all of these obstacles are a source of replication stress. Replication stress is one of the main hallmarks of cancer and its prevention is becoming increasingly important as a target for future chemotherapeutics. Here we will try to summarize how these three obstacles are generated and how the cells handle replication stress upon their encounter. Finally, we will consider their role in cancer and their exploitation in current chemotherapeutic approaches.

Phosphorylation regulates human polη stability and damage bypass throughout the cell cycle.

DNA translesion synthesis (TLS) is a crucial damage tolerance pathway that oversees the completion of DNA replication in the presence of DNA damage. TLS polymerases are capable of bypassing a distorted template but they are generally considered inaccurate and they need to be tightly regulated. We have previously shown that polη is phosphorylated on Serine 601 after DNA damage and we have demonstrated that this modification is important for efficient damage bypass. Here we report that polη is also phosphorylated by CDK2, in the absence of damage, in a cell cycle-dependent manner and we identify serine 687 as an important residue targeted by the kinase. We discover that phosphorylation on serine 687 regulates the stability of the polymerase during the cell cycle, allowing it to accumulate in late S and G2 when productive TLS is critical for cell survival. Furthermore, we show that alongside the phosphorylation of S601, the phosphorylation of S687 and S510, S512 and/or S514 are important for damage bypass and cell survival after UV irradiation. Taken together our results provide new insights into how cells can, at different times, modulate DNA TLS for improved cell survival.

Ribonucleotide incorporation by human DNA polymerase η impacts translesion synthesis and RNase H2 activity.

Ribonucleotides (rNs) incorporated in the genome by DNA polymerases (Pols) are removed by RNase H2. Cytidine and guanosine preferentially accumulate over the other rNs. Here we show that human Pol η can incorporate cytidine monophosphate (rCMP) opposite guanine, 8-oxo-7,8-dihydroguanine, 8-methyl-2΄-deoxyguanosine and a cisplatin intrastrand guanine crosslink (cis-PtGG), while it cannot bypass a 3-methylcytidine or an abasic site with rNs as substrates. Pol η is also capable of synthesizing polyribonucleotide chains, and its activity is enhanced by its auxiliary factor DNA Pol δ interacting protein 2 (PolDIP2). Human RNase H2 removes cytidine and guanosine less efficiently than the other rNs and incorporation of rCMP opposite DNA lesions further reduces the efficiency of RNase H2. Experiments with XP-V cell extracts indicate Pol η as the major basis of rCMP incorporation opposite cis-PtGG. These results suggest that translesion synthesis by Pol η can contribute to the accumulation of rCMP in the genome, particularly opposite modified guanines.

4-PBA ameliorates cellular homeostasis in fibroblasts from osteogenesis imperfecta patients by enhancing autophagy and stimulating protein secretion.

The clinical phenotype in osteogenesis imperfecta (OI) is attributed to the dominant negative function of mutant type I collagen molecules in the extracellular matrix, by altering its structure and function. Intracellular retention of mutant collagen has also been reported, but its effect on cellular homeostasis is less characterized. Using OI patient fibroblasts carrying mutations in the α1(I) and α2(I) chains we demonstrate that retained collagen molecules are responsible for endoplasmic reticulum (ER) enlargement and activation of the unfolded protein response (UPR) mainly through the eukaryotic translation initiation factor 2 alpha kinase 3 (PERK) branch. Cells carrying α1(I) mutations upregulate autophagy, while cells with α2(I) mutations only occasionally activate the autodegradative response. Despite the autophagy activation to face stress conditions, apoptosis occurs in all mutant fibroblasts. To reduce cellular stress, mutant fibroblasts were treated with the FDA-approved chemical chaperone 4-phenylbutyric acid. The drug rescues cell death by modulating UPR activation thanks to both its chaperone and histone deacetylase inhibitor abilities. As chaperone it increases general cellular protein secretion in all patients' cells as well as collagen secretion in cells with the most C-terminal mutation. As histone deacetylase inhibitor it enhances the expression of the autophagic gene Atg5 with a consequent stimulation of autophagy. These results demonstrate that the cellular response to ER stress can be a relevant target to ameliorate OI cell homeostasis.

Regulation of translesion synthesis DNA polymerase eta by monoubiquitination.

DNA polymerase eta is a Y family polymerase involved in translesion synthesis (TLS). Its action is initiated by simultaneous interaction between the PIP box in pol eta and PCNA and between the UBZ in pol eta and monoubiquitin attached to PCNA. Whereas monoubiquitination of PCNA is required for its interaction with pol eta during TLS, we now show that monoubiquitination of pol eta inhibits this interaction, preventing its functions in undamaged cells. Identification of monoubiquitination sites within pol eta nuclear localization signal (NLS) led to the discovery that pol eta NLS directly contacts PCNA, forming an extended pol eta-PCNA interaction surface. We name this the PCNA-interacting region (PIR) and show that its monoubiquitination is downregulated by various DNA-damaging agents. We propose that this mechanism ensures optimal availability of nonubiquitinated, TLS-competent pol eta after DNA damage. Our work shows how monoubiquitination can either positively or negatively regulate the assembly of a protein complex, depending on which substrates are targeted by ubiquitin.

Homozygous substitution of threonine 191 by proline in polymerase η causes Xeroderma pigmentosum variant.

DNA polymerase eta (Polη) is the only translesion synthesis polymerase capable of error-free bypass of UV-induced cyclobutane pyrimidine dimers. A deficiency in Polη function is associated with the human disease Xeroderma pigmentosum variant (XPV). We hereby report the case of a 60-year-old woman known for XPV and carrying a Polη Thr191Pro variant in homozygosity. We further characterize the variant in vitro and in vivo, providing molecular evidence that the substitution abrogates polymerase activity and results in UV sensitivity through deficient damage bypass. This is the first functional molecular characterization of a missense variant of Polη, whose reported pathogenic variants have thus far been loss of function truncation or frameshift mutations. Our work allows the upgrading of Polη Thr191Pro from 'variant of uncertain significance' to 'likely pathogenic mutant', bearing direct impact on molecular diagnosis and genetic counseling. Furthermore, we have established a robust experimental approach that will allow a precise molecular analysis of further missense mutations possibly linked to XPV. Finally, it provides insight into critical Polη residues that may be targeted to develop small molecule inhibitors for cancer therapeutics.

HDAC6 inhibition disrupts HDAC6-P300 interaction reshaping the cancer chromatin landscape.

BACKGROUND: Histone deacetylases (HDACs) are crucial regulators of gene expression, DNA synthesis, and cellular processes, making them essential targets in cancer research. HDAC6, specifically, influences protein stability and chromatin dynamics. Despite HDAC6's potential therapeutic value, its exact role in gene regulation and chromatin remodeling needs further clarification. This study examines how HDAC6 inactivation influences lysine acetyltransferase P300 stabilization and subsequent effects on chromatin structure and function in cancer cells. METHODS AND RESULTS: We employed the HDAC6 inhibitor ITF3756, siRNA, or CRISPR/Cas9 gene editing to inactivate HDAC6 in different epigenomic backgrounds. Constantly, this inactivation led to significant changes in chromatin accessibility, particularly increased acetylation of histone H3 lysines 9, 14, and 27 (ATAC-seq and H3K27Ac ChIP-seq analysis). Transcriptomics, proteomics, and gene ontology analysis revealed gene changes in cell proliferation, adhesion, migration, and apoptosis. Significantly, HDAC6 inactivation altered P300 ubiquitination, stabilizing P300 and leading to downregulating genes critical for cancer cell survival. CONCLUSIONS: Our study highlights the substantial impact of HDAC6 inactivation on the chromatin landscape of cancer cells and suggests a role for P300 in contributing to the anticancer effects. The stabilization of P300 with HDAC6 inhibition proposes a potential shift in therapeutic focus from HDAC6 itself to its interaction with P300. This finding opens new avenues for developing targeted cancer therapies, improving our understanding of epigenetic mechanisms in cancer cells.

Obesogenic High-Fat Diet and MYC Cooperate to Promote Lactate Accumulation and Tumor Microenvironment Remodeling in Prostate Cancer.

Cancer cells exhibit metabolic plasticity to meet oncogene-driven dependencies while coping with nutrient availability. A better understanding of how systemic metabolism impacts the accumulation of metabolites that reprogram the tumor microenvironment (TME) and drive cancer could facilitate development of precision nutrition approaches. Using the Hi-MYC prostate cancer mouse model, we demonstrated that an obesogenic high-fat diet (HFD) rich in saturated fats accelerates the development of c-MYC-driven invasive prostate cancer through metabolic rewiring. Although c-MYC modulated key metabolic pathways, interaction with an obesogenic HFD was necessary to induce glycolysis and lactate accumulation in tumors. These metabolic changes were associated with augmented infiltration of CD206+ and PD-L1+ tumor-associated macrophages (TAM) and FOXP3+ regulatory T cells, as well as with the activation of transcriptional programs linked to disease progression and therapy resistance. Lactate itself also stimulated neoangiogenesis and prostate cancer cell migration, which were significantly reduced following treatment with the lactate dehydrogenase inhibitor FX11. In patients with prostate cancer, high saturated fat intake and increased body mass index were associated with tumor glycolytic features that promote the infiltration of M2-like TAMs. Finally, upregulation of lactate dehydrogenase, indicative of a lactagenic phenotype, was associated with a shorter time to biochemical recurrence in independent clinical cohorts. This work identifies cooperation between genetic drivers and systemic metabolism to hijack the TME and promote prostate cancer progression through oncometabolite accumulation. This sets the stage for the assessment of lactate as a prognostic biomarker and supports strategies of dietary intervention and direct lactagenesis blockade in treating advanced prostate cancer. SIGNIFICANCE: Lactate accumulation driven by high-fat diet and MYC reprograms the tumor microenvironment and promotes prostate cancer progression, supporting the potential of lactate as a biomarker and therapeutic target in prostate cancer. See related commentary by Frigo, p. 1742.

Sex specific regulation of TSPY-Like 2 in the DNA damage response of cancer cells.

Females have a lower probability to develop somatic cancers and a better response to chemotherapy than males. However, the reasons for these differences are still not well understood. The X-linked gene TSPY-Like 2 (TSPYL2) encodes for a putative tumor suppressor protein involved in cell cycle regulation and DNA damage response (DDR) pathways. Here, we demonstrate that in unstressed conditions TSPYL2 is maintained at low levels by MDM2-dependent ubiquitination and proteasome degradation. Upon genotoxic stress, E2F1 promotes TSPYL2 expression and protein accumulation in non-transformed cell lines. Conversely, in cancer cells, TSPYL2 accumulates only in females or in those male cancer cells that lost the Y-chromosome during the oncogenic process. Hence, we demonstrate that while TSPYL2 mRNA is induced in all the tested tumor cell lines after DNA damage, TSPYL2 protein stability is increased only in female cancer cells. Indeed, we found that TSPYL2 accumulation, in male cancer cells, is prevented by the Y-encoded protein SRY, which modulates MDM2 protein levels. In addition, we demonstrated that TSPYL2 accumulation is required to sustain cell growth arrest after DNA damage, possibly contributing to protect normal and female cancer cells from tumor progression. Accordingly, TSPYL2 has been found more frequently mutated in female-specific cancers. These findings demonstrate for the first time a sex-specific regulation of TSPYL2 in the DDR of cancer cells and confirm the existence of sexual dimorphism in DNA surveillance pathways.

Regulation of proliferating cell nuclear antigen ubiquitination in mammalian cells.

After exposure to DNA-damaging agents that block the progress of the replication fork, monoubiquitination of proliferating cell nuclear antigen (PCNA) mediates the switch from replicative to translesion synthesis DNA polymerases. We show that in human cells, PCNA is monoubiquitinated in response to methyl methanesulfonate and mitomycin C, as well as UV light, albeit with different kinetics, but not in response to bleomycin or camptothecin. Cyclobutane pyrimidine dimers are responsible for most of the PCNA ubiquitination events after UV-irradiation. Failure to ubiquitinate PCNA results in substantial sensitivity to UV and methyl methanesulfonate, but not to camptothecin or bleomycin. PCNA ubiquitination depends on Replication Protein A (RPA), but is independent of ATR-mediated checkpoint activation. After UV-irradiation, there is a temporal correlation between the disappearance of the deubiquitinating enzyme USP1 and the presence of PCNA ubiquitination, but this correlation was not found after chemical mutagen treatment. By using cells expressing photolyases, we are able to remove the UV lesions, and we show that PCNA ubiquitination persists for many hours after the damage has been removed. We present a model of translesion synthesis behind the replication fork to explain the persistence of ubiquitinated PCNA.

Cellular stress due to impairment of collagen prolyl hydroxylation complex is rescued by the chaperone 4-phenylbutyrate.

Osteogenesis imperfecta (OI) types VII, VIII and IX, caused by recessive mutations in cartilage-associated protein (CRTAP), prolyl-3-hydroxylase 1 (P3H1) and cyclophilin B (PPIB), respectively, are characterized by the synthesis of overmodified collagen. The genes encode for the components of the endoplasmic reticulum (ER) complex responsible for the 3-hydroxylation of specific proline residues in type I collagen. Our study dissects the effects of mutations in the proteins of the complex on cellular homeostasis, using primary fibroblasts from seven recessive OI patients. In all cell lines, the intracellular retention of overmodified type I collagen molecules causes ER enlargement associated with the presence of protein aggregates, activation of the PERK branch of the unfolded protein response and apoptotic death. The administration of 4-phenylbutyrate (4-PBA) alleviates cellular stress by restoring ER cisternae size, and normalizing the phosphorylated PERK (p-PERK):PERK ratio and the expression of apoptotic marker. The drug also has a stimulatory effect on autophagy. We proved that the rescue of cellular homeostasis following 4-PBA treatment is associated with its chaperone activity, since it increases protein secretion, restoring ER proteostasis and reducing PERK activation and cell survival also in the presence of pharmacological inhibition of autophagy. Our results provide a novel insight into the mechanism of 4-PBA action and demonstrate that intracellular stress in recessive OI can be alleviated by 4-PBA therapy, similarly to what we recently reported for dominant OI, thus allowing a common target for OI forms characterized by overmodified collagen.This article has an associated First Person interview with the first author of the paper.

Gene Expression Profiles Controlled by the Alternative Splicing Factor Nova2 in Endothelial Cells.

Alternative splicing (AS) plays an important role in expanding the complexity of the human genome through the production of specialized proteins regulating organ development and physiological functions, as well as contributing to several pathological conditions. How AS programs impact on the signaling pathways controlling endothelial cell (EC) functions and vascular development is largely unknown. Here we identified, through RNA-seq, changes in mRNA steady-state levels in ECs caused by the neuro-oncological ventral antigen 2 (Nova2), a key AS regulator of the vascular morphogenesis. Bioinformatics analyses identified significant enrichment for genes regulated by peroxisome proliferator-activated receptor-gamma (Ppar-γ) and E2F1 transcription factors. We also showed that Nova2 in ECs controlled the AS profiles of Ppar-γ and E2F dimerization partner 2 (Tfdp2), thus generating different protein isoforms with distinct function (Ppar-γ) or subcellular localization (Tfdp2). Collectively, our results supported a mechanism whereby Nova2 integrated splicing decisions in order to regulate Ppar-γ and E2F1 activities. Our data added a layer to the sequential series of events controlled by Nova2 in ECs to orchestrate vascular biology.

Yeast Rev1 is cell cycle regulated, phosphorylated in response to DNA damage and its binding to chromosomes is dependent upon MEC1.

Translesion DNA synthesis (TLS) is one of the mechanisms involved in lesion bypass during DNA replication. Three TLS polymerases (Pol) are present in the yeast Saccharomyces cerevisiae: Pol zeta, Pol eta and the product of the REV1 gene. Rev1 is considered a deoxycytidyl transferase because it almost exclusively inserts a C residue in front of the lesion. Even though REV1 is required for most of the UV-induced and spontaneous mutagenesis events, the role of Rev1 is poorly understood since its polymerase activity is often dispensable. Rev1 interacts with several TLS polymerases in mammalian cells and may act as a platform in the switching mechanism required to substitute a replicative polymerase with a TLS polymerase at the sites of DNA lesions. Here we show that yeast Rev1 is a phosphoprotein, and the level of this modification is cell cycle regulated under normal growing conditions. Rev1 is unphosphorylated in G1, starts to be modified while cells are passing S phase and it becomes hyper-phosphorylated in mitosis. Rev1 is also hyper-phosphorylated in response to a variety of DNA damaging agents, including treatment with a radiomimetic drug mostly causing double-strand breaks (DSB). By using the chromosome spreading technique we found the Rev1 is bound to chromosomes throughout the cell cycle, and its binding does not significantly increase in response to genotoxic stress. Therefore, Rev1 phosphorylation does not appear to modulate its binding to chromosomes, suggesting that such modification may influence other aspects of the TLS process. Rev1 binding under damaged and undamaged conditions, is at least partially dependent on MEC1, a gene playing a pivotal role in the DNA damage checkpoint cascade. This genetic dependency may suggest a role for MEC1 in spontaneous mutagenesis events, which require a functional REV1 gene.

Translesion synthesis: Y-family polymerases and the polymerase switch.

Replicative DNA polymerases are blocked at DNA lesions. Synthesis past DNA damage requires the replacement of the replicative polymerase by one of a group of specialised translesion synthesis (TLS) polymerases, most of which belong to the Y-family. Each of these has different substrate specificities for different types of damage. In eukaryotes mono-ubiquitination of PCNA plays a crucial role in the switch from replicative to TLS polymerases at stalled forks. All the Y-family polymerases have ubiquitin binding sites that increase their binding affinity for ubiquitinated PCNA at the sites of stalled forks.