Checkpoint-mediated DNA polymerase ε exonuclease activity curbing counteracts resection-driven fork collapse.

DNA polymerase ε (Polε) carries out high-fidelity leading strand synthesis owing to its exonuclease activity. Polε polymerase and exonuclease activities are balanced, because of partitioning of nascent DNA strands between catalytic sites, so that net resection occurs when synthesis is impaired. In vivo, DNA synthesis stalling activates replication checkpoint kinases, which act to preserve the functional integrity of replication forks. We show that stalled Polε drives nascent strand resection causing fork functional collapse, averted via checkpoint-dependent phosphorylation. Polε catalytic subunit Pol2 is phosphorylated on serine 430, influencing partitioning between polymerase and exonuclease active sites. A phosphormimetic S430D change reduces exonucleolysis in vitro and counteracts fork collapse. Conversely, non-phosphorylatable pol2-S430A expression causes resection-driven stressed fork defects. Our findings reveal that checkpoint kinases switch Polε to an exonuclease-safe mode preventing nascent strand resection and stabilizing stalled replication forks. Elective partitioning suppression has implications for the diverse Polε roles in genome integrity maintenance.

Senataxin associates with replication forks to protect fork integrity across RNA-polymerase-II-transcribed genes.

Transcription hinders replication fork progression and stability. The ATR checkpoint and specialized DNA helicases assist DNA synthesis across transcription units to protect genome integrity. Combining genomic and genetic approaches together with the analysis of replication intermediates, we searched for factors coordinating replication with transcription. We show that the Sen1/Senataxin DNA/RNA helicase associates with forks, promoting their progression across RNA polymerase II (RNAPII)-transcribed genes. sen1 mutants accumulate aberrant DNA structures and DNA-RNA hybrids while forks clash head-on with RNAPII transcription units. These replication defects correlate with hyperrecombination and checkpoint activation in sen1 mutants. The Sen1 function at the forks is separable from its role in RNA processing. Our data, besides unmasking a key role for Senataxin in coordinating replication with transcription, provide a framework for understanding the pathological mechanisms caused by Senataxin deficiencies and leading to the severe neurodegenerative diseases ataxia with oculomotor apraxia type 2 and amyotrophic lateral sclerosis 4.

The replication checkpoint protects fork stability by releasing transcribed genes from nuclear pores.

Transcription hinders replication fork progression and stability, and the Mec1/ATR checkpoint protects fork integrity. Examining checkpoint-dependent mechanisms controlling fork stability, we find that fork reversal and dormant origin firing due to checkpoint defects are rescued in checkpoint mutants lacking THO, TREX-2, or inner-basket nucleoporins. Gene gating tethers transcribed genes to the nuclear periphery and is counteracted by checkpoint kinases through phosphorylation of nucleoporins such as Mlp1. Checkpoint mutants fail to detach transcribed genes from nuclear pores, thus generating topological impediments for incoming forks. Releasing this topological complexity by introducing a double-strand break between a fork and a transcribed unit prevents fork collapse. Mlp1 mutants mimicking constitutive checkpoint-dependent phosphorylation also alleviate checkpoint defects. We propose that the checkpoint assists fork progression and stability at transcribed genes by phosphorylating key nucleoporins and counteracting gene gating, thus neutralizing the topological tension generated at nuclear pore gated genes.

Cohesin Ubiquitylation and Mobilization Facilitate Stalled Replication Fork Dynamics.

Replication fork integrity is challenged in conditions of stress and protected by the Mec1/ATR checkpoint to preserve genome stability. Still poorly understood in fork protection is the role played by the structural maintenance of chromosomes (SMC) cohesin complex. We uncovered a role for the Rsp5Bul2 ubiquitin ligase in promoting survival to replication stress by preserving stalled fork integrity. Rsp5Bul2 physically interacts with cohesin and the Mec1 kinase, thus promoting checkpoint-dependent cohesin ubiquitylation and cohesin-mediated fork protection. Ubiquitylation mediated by Rsp5Bul2 promotes cohesin mobilization from chromatin neighboring stalled forks, likely by stimulating the Cdc48/p97 ubiquitin-selective segregase, and its timely association to nascent chromatids. This Rsp5Bul2 fork protection mechanism requires the Wpl1 cohesin mobilizer as well as the function of the Eco1 acetyltransferase securing sister chromatid entrapment. Our data indicate that ubiquitylation facilitates cohesin dynamic interfacing with replication forks within a mechanism preserving stalled-fork functional architecture.

PP2A Controls Genome Integrity by Integrating Nutrient-Sensing and Metabolic Pathways with the DNA Damage Response.

Mec1ATR mediates the DNA damage response (DDR), integrating chromosomal signals and mechanical stimuli. We show that the PP2A phosphatases, ceramide-activated enzymes, couple cell metabolism with the DDR. Using genomic screens, metabolic analysis, and genetic and pharmacological studies, we found that PP2A attenuates the DDR and that three metabolic circuits influence the DDR by modulating PP2A activity. Irc21, a putative cytochrome b5 reductase that promotes the condensation reaction generating dihydroceramides (DHCs), and Ppm1, a PP2A methyltransferase, counteract the DDR by activating PP2A; conversely, the nutrient-sensing TORC1-Tap42 axis sustains DDR activation by inhibiting PP2A. Loss-of-function mutations in IRC21, PPM1, and PP2A and hyperactive tap42 alleles rescue mec1 mutants. Ceramides synergize with rapamycin, a TORC1 inhibitor, in counteracting the DDR. Hence, PP2A integrates nutrient-sensing and metabolic pathways to attenuate the Mec1ATR response. Our observations imply that metabolic changes affect genome integrity and may help with exploiting therapeutic options and repositioning known drugs.

Replicon dynamics, dormant origin firing, and terminal fork integrity after double-strand break formation.

In response to replication stress, the Mec1/ATR and SUMO pathways control stalled- and damaged-fork stability. We investigated the S phase response at forks encountering a broken template (termed the terminal fork). We show that double-strand break (DSB) formation can locally trigger dormant origin firing. Irreversible fork resolution at the break does not impede progression of the other fork in the same replicon (termed the sister fork). The Mre11-Tel1/ATM response acts at terminal forks, preventing accumulation of cruciform DNA intermediates that tether sister chromatids and can undergo nucleolytic processing. We conclude that sister forks can be uncoupled during replication and that, after DSB-induced fork termination, replication is rescued by dormant origin firing or adjacent replicons. We have uncovered a Tel1/ATM- and Mre11-dependent response controlling terminal fork integrity. Our findings have implications for those genome instability syndromes that accumulate DNA breaks during S phase and for forks encountering eroding telomeres.

Genome-organizing factors Top2 and Hmo1 prevent chromosome fragility at sites of S phase transcription.

Specialized topoisomerases solve the topological constraints arising when replication forks encounter transcription. We have investigated the contribution of Top2 in S phase transcription. Specifically in S phase, Top2 binds intergenic regions close to transcribed genes. The Top2-bound loci exhibit low nucleosome density and accumulate gammaH2A when Top2 is defective. These intergenic loci associate with the HMG protein Hmo1 throughout the cell cycle and are refractory to the histone variant Htz1. In top2 mutants, Hmo1 is deleterious and accumulates at pericentromeric regions in G2/M. Our data indicate that Top2 is dispensable for transcription and that Hmo1 and Top2 bind in the proximity of genes transcribed in S phase suppressing chromosome fragility at the M-G1 transition. We propose that an Hmo1-dependent epigenetic signature together with Top2 mediate an S phase architectural pathway to preserve genome integrity.

Cyba-deficient mice display an increase in hematopoietic stem cells and an overproduction of immunoglobulins.

The regulation of protein function by reversible oxidation is increasingly recognized as a key mechanism for the control of cellular signaling, modulating crucial biological processes such as cell differentiation. In this scenario, NADPH oxidases must occupy a prominent position. Our results show that hematopoietic stem and progenitor cells express three p22phox-dependent NADPH oxidases members (NOX1, NOX2 and NOX4). By deleting the p22phox coding gene (Cyba), here we have analyzed the importance of this family of enzymes during in vivo hematopoiesis. Cyba-/- mice show a myeloid bias, and an enrichment of hematopoietic stem cell populations. By means of hematopoietic transplant experiments we have also tried to dissect the specific role of the NADPH oxidases. While the absence of NOX1 or NOX2 provides a higher level of reconstitution, a lack of NOX4 rendered the opposite result, suggesting a functional specificity among the different NADPH oxidases. Cyba-/- cells showed a hampered activation of AKT1 and a sharp decrease in STAT5 protein. This is in line with the diminished response to IL-7 shown by our results, which could explain the overproduction of immunoglobulins observed in Cyba-/- mice.

Granuloma Formation in a Cyba-Deficient Model of Chronic Granulomatous Disease Is Associated with Myeloid Hyperplasia and the Exhaustion of B-Cell Lineage.

Haematopoiesis is a paradigm of cell differentiation because of the wide variety and overwhelming number of mature blood cells produced daily. Under stress conditions, the organism must adapt to a boosted demand for blood cells. Chronic granulomatous disease (CGD) is a genetic disease caused by inactivating mutations that affect the phagocyte oxidase. Besides a defective innate immune system, CGD patients suffer from recurrent hyper-inflammation episodes, circumstances upon which they must face emergency haematopoiesis. The targeting of Cybb and Ncf1 genes have produced CGD animal models that are a useful surrogate when studying the pathophysiology and treatment of this disease. Here, we show that Cyba-/- mice spontaneously develop granuloma and, therefore, constitute a CGD animal model to complement the existing Cybb-/- and Ncf1-/- models. More importantly, we have analysed haematopoiesis in granuloma-bearing Cyba-/- mice. These animals showed a significant loss of weight, developed remarkable splenomegaly, bone marrow myeloid hyperplasia, and signs of anaemia. Haematological analyses showed a sharped decrease of B-cells and a striking development of myeloid cells in all compartments. Collectively, our results show that granuloma inflammatory lesions dramatically change haematopoiesis homeostasis. Consequently, we suggest that besides their defective innate immunity, the alteration of haematopoiesis homeostasis upon granuloma may contribute to the dismal outcome of CGD.

Preventing replication stress to maintain genome stability: resolving conflicts between replication and transcription.

DNA and RNA polymerases clash along the genome as they compete for the same DNA template. Cells have evolved specialized strategies to prevent and resolve replication and transcription interference. Here, we review the topology and architecture at sites of replication fork clashes with transcription bubbles as well as the regulatory circuits that control replication fork passage across transcribed genes. In the case of RNA polymerase II-transcribed genes, cotranscriptional processes such as mRNA maturation, splicing, and export influence the integrity of replication forks and transcribed loci. Fork passage likely contributes to reset the epigenetic landscape, influencing gene expression and transcriptional memory. When any of these processes are not properly coordinated, aberrant outcomes such as fork reversal and R-loop formation arise and trigger unscheduled recombinogenic events and genome rearrangements. The evolutionary implications of such conflicts on genome dynamics and their potential impact on oncogenic stress are discussed.

Replisome-Cohesin Interfacing: A Molecular Perspective.

Cohesion is established in S-phase through the action of key replisome factors as replication forks engage cohesin molecules. By holding sister chromatids together, cohesion critically assists both an equal segregation of the duplicated genetic material and an efficient repair of DNA breaks. Nonetheless, the molecular events leading the entrapment of nascent chromatids by cohesin during replication are only beginning to be understood. The authors describe here the essential structural features of the cohesin complex in connection to its ability to associate DNA molecules and review the current knowledge on the architectural-functional organization of the eukaryotic replisome, significantly advanced by recent biochemical and structural studies. In light of this novel insight, the authors discuss the mechanisms proposed to assist interfacing of replisomes with chromatin-bound cohesin complexes and elaborate on models for nascent chromatids entrapment by cohesin in the environment of the replication fork.

DNA bending facilitates the error-free DNA damage tolerance pathway and upholds genome integrity.

DNA replication is sensitive to damage in the template. To bypass lesions and complete replication, cells activate recombination-mediated (error-free) and translesion synthesis-mediated (error-prone) DNA damage tolerance pathways. Crucial for error-free DNA damage tolerance is template switching, which depends on the formation and resolution of damage-bypass intermediates consisting of sister chromatid junctions. Here we show that a chromatin architectural pathway involving the high mobility group box protein Hmo1 channels replication-associated lesions into the error-free DNA damage tolerance pathway mediated by Rad5 and PCNA polyubiquitylation, while preventing mutagenic bypass and toxic recombination. In the process of template switching, Hmo1 also promotes sister chromatid junction formation predominantly during replication. Its C-terminal tail, implicated in chromatin bending, facilitates the formation of catenations/hemicatenations and mediates the roles of Hmo1 in DNA damage tolerance pathway choice and sister chromatid junction formation. Together, the results suggest that replication-associated topological changes involving the molecular DNA bender, Hmo1, set the stage for dedicated repair reactions that limit errors during replication and impact on genome stability.

Cohesin dynamic association to chromatin and interfacing with replication forks in genome integrity maintenance.

Proliferating cells need to accurately duplicate and pass their genetic material on to daughter cells. Problems during replication and partition challenge the structural and numerical integrity of chromosomes. Diverse mechanisms, as the DNA replication checkpoint, survey the correct progression of replication and couple it with other cell cycle events to preserve genome integrity. The structural maintenance of chromosomes (SMC) cohesin complex primarily contributes to chromosome duplication by mediating the tethering of newly replicated sister chromatids, thus assisting their equal segregation in mitosis. In addition, cohesin exerts important functions in genome organization, gene expression and DNA repair. These are determined by cohesin's ability to bring together different DNA segments and, hence, by the fashion and dynamics of its interaction with chromatin. It recently emerged that cohesin contributes to the protection of stalled replication forks through a mechanism requiring its timely mobilization from unreplicated DNA and relocation to nascent strands. This mechanism relies on DNA replication checkpoint-dependent cohesin ubiquitylation and promotes nascent sister chromatid entrapment, likely contributing to preserve stalled replisome-fork architectural integrity. Here we review how cohesin dynamic association to chromatin is controlled through post-translational modifications to dictate its functions during chromosome duplication. We also discuss recent insights on the mechanism that mediates interfacing of replisome components with chromatin-bound cohesin and its contribution to the establishment of sister chromatid cohesion and the protection of stalled replication forks.

Replication termination at eukaryotic chromosomes is mediated by Top2 and occurs at genomic loci containing pausing elements.

Chromosome replication initiates at multiple replicons and terminates when forks converge. In E. coli, the Tus-TER complex mediates polar fork converging at the terminator region, and aberrant termination events challenge chromosome integrity and segregation. Since in eukaryotes, termination is less characterized, we used budding yeast to identify the factors assisting fork fusion at replicating chromosomes. Using genomic and mechanistic studies, we have identified and characterized 71 chromosomal termination regions (TERs). TERs contain fork pausing elements that influence fork progression and merging. The Rrm3 DNA helicase assists fork progression across TERs, counteracting the accumulation of X-shaped structures. The Top2 DNA topoisomerase associates at TERs in S phase, and G2/M facilitates fork fusion and prevents DNA breaks and genome rearrangements at TERs. We propose that in eukaryotes, replication fork barriers, Rrm3, and Top2 coordinate replication fork progression and fusion at TERs, thus counteracting abnormal genomic transitions.

Nucleolytic processing of aberrant replication intermediates by an Exo1-Dna2-Sae2 axis counteracts fork collapse-driven chromosome instability.

Problems during DNA replication underlie genomic instability and drive malignant transformation. The DNA damage checkpoint stabilizes stalled replication forks thus counteracting aberrant fork transitions, DNA breaks and chromosomal rearrangements. We analyzed fork processing in checkpoint deficient cells by coupling psoralen crosslinking with replication intermediate two-dimensional gel analysis. This revealed a novel role for Exo1 nuclease in resecting reversed replication fork structures and counteracting the accumulation of aberrant intermediates resembling fork cleavage products. Genetic analyses demonstrated a functional interplay of Exo1 with Mus81, Dna2 and Sae2 nucleases in promoting cell survival following replication stress, suggestive of concerted nucleolytic processing of stalled forks. While Mus81 and other Structure Specific Endonucleases do not contribute to obvious collapsed fork transitions, Dna2 promotes reversed fork resection likely by facilitating Exo1 access to nascent strands. Instead, Sae2 cooperates with Exo1 in counteracting putative fork cleavage events linked to double strand breaks formation and increased gross chromosomal rearrangement rates. Our data indicate that in checkpoint deficient cells diverse nuclease activities interface to eliminate aberrant replication intermediates and prevent chromosome instability.

The Multiple Roles of Ubiquitylation in Regulating Challenged DNA Replication.

DNA replication is essential for the propagation of life and the development of complex organisms. However, replication is a risky process as it can lead to mutations and chromosomal alterations. Conditions challenging DNA synthesis by replicative polymerases or DNA helix unwinding, generally termed as replication stress, can halt replication fork progression. Stalled replication forks are unstable, and mechanisms exist to protect their integrity, which promote an efficient restart of DNA synthesis and counteract fork collapse characterized by the accumulation of DNA lesions and mutagenic events. DNA replication is a highly regulated process, and several mechanisms control replication timing and integrity both during unperturbed cell cycles and in response to replication stress. Work over the last two decades has revealed that key steps of DNA replication are controlled by conjugation of the small peptide ubiquitin. While ubiquitylation was traditionally linked to protein degradation, the complexity and flexibility of the ubiquitin system in regulating protein function have recently emerged. Here we review the multiple roles exerted by ubiquitin-conjugating enzymes and ubiquitin-specific proteases, as well as readers of ubiquitin chains, in the control of eukaryotic DNA replication and replication-coupled DNA damage tolerance and repair.

PTPN13 regulates cellular signalling and ß-catenin function during megakaryocytic differentiation.

PTPN13 is a high-molecular weight intracellular phosphatase with several isoforms that exhibits a highly modular structure. Although in recent years different roles have been described for PTPN13, we are still far from understanding its function in cell biology. Here we show that PTPN13 expression is activated during megakaryocytic differentiation at the protein and mRNA level. Our results show that the upregulation of PTPN13 inhibits megakaryocytic differentiation, while PTPN13 silencing triggers differentiation. The ability of PTPN13 to alter megakaryocytic differentiation can be explained by its capacity to regulate ERK and STAT signalling. Interestingly, the silencing of ß-catenin produced the same effect as PTPN13 downregulation. We demonstrate that both proteins coimmunoprecipitate and colocalise. Moreover, we provide evidence showing that PTPN13 can regulate ß-catenin phosphorylation, stability and transcriptional activity. Therefore, the ability of PTPN13 to control megakaryocytic differentiation must be intimately linked to the regulation of ß-catenin function. Moreover, our results show for the first time that PTPN13 is stabilised upon Wnt signalling, which makes PTPN13 an important player in canonical Wnt signalling. Our results show that PTPN13 behaves as an important regulator of megakaryocytic differentiation in cell lines and also in murine haematopoietic progenitors. This importance can be explained by the ability of PTPN13 to regulate cellular signalling, and especially through the regulation of ß-catenin stability and function. Our results hold true for different megakaryocytic cell lines and also for haematopoietic progenitors, suggesting that these two proteins may play a relevant role during in vivo megakaryopoiesis.

Genome-wide function of THO/TREX in active genes prevents R-loop-dependent replication obstacles.

THO/TREX is a conserved nuclear complex that functions in mRNP biogenesis and prevents transcription-associated recombination. Whether or not it has a ubiquitous role in the genome is unknown. Chromatin immunoprecipitation (ChIP)-chip studies reveal that the Hpr1 component of THO and the Sub2 RNA-dependent ATPase have genome-wide distributions at active ORFs in yeast. In contrast to RNA polymerase II, evenly distributed from promoter to termination regions, THO and Sub2 are absent at promoters and distributed in a gradual 5' → 3' gradient. This is accompanied by a genome-wide impact of THO-Sub2 deletions on expression of highly expressed, long and high G+C-content genes. Importantly, ChIP-chips reveal an over-recruitment of Rrm3 in active genes in THO mutants that is reduced by RNaseH1 overexpression. Our work establishes a genome-wide function for THO-Sub2 in transcription elongation and mRNP biogenesis that function to prevent the accumulation of transcription-mediated replication obstacles, including R-loops.

G-quadruplex-induced instability during leading-strand replication.

G-quadruplexes are four-stranded nucleic acid structures whose biological functions remain poorly understood. In the yeast S. cerevisiae, we report that G-quadruplexes form and, if not properly processed, pose a specific challenge to replication. We show that the G-quadruplex-prone CEB1 tandem array is tolerated when inserted near ARS305 replication origin in wild-type cells but is very frequently destabilized upon treatment with the potent Phen-DC(3) G-quadruplex ligand, or in the absence of the G-quadruplex-unwinding Pif1 helicase, only when the G-rich strand is the template of leading-strand replication. The orientation-dependent instability is associated with the formation of Rad51-Rad52-dependent X-shaped intermediates during replication detected by two-dimensional (2D) gels, and relies on the presence of intact G-quadruplex motifs in CEB1 and on the activity of ARS305. The asymmetrical behaviour of G-quadruplex prone sequences during replication has implications for their evolutionary dynamics within genomes, including the maintenance of G-rich telomeres.

Unlocking the power of AI models: exploring protein folding prediction through comparative analysis.

Protein structure determination has made progress with the aid of deep learning models, enabling the prediction of protein folding from protein sequences. However, obtaining accurate predictions becomes essential in certain cases where the protein structure remains undescribed. This is particularly challenging when dealing with rare, diverse structures and complex sample preparation. Different metrics assess prediction reliability and offer insights into result strength, providing a comprehensive understanding of protein structure by combining different models. In a previous study, two proteins named ARM58 and ARM56 were investigated. These proteins contain four domains of unknown function and are present in Leishmania spp. ARM refers to an antimony resistance marker. The study's main objective is to assess the accuracy of the model's predictions, thereby providing insights into the complexities and supporting metrics underlying these findings. The analysis also extends to the comparison of predictions obtained from other species and organisms. Notably, one of these proteins shares an ortholog with Trypanosoma cruzi and Trypanosoma brucei, leading further significance to our analysis. This attempt underscored the importance of evaluating the diverse outputs from deep learning models, facilitating comparisons across different organisms and proteins. This becomes particularly pertinent in cases where no previous structural information is available.