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Biallelic loss of cyclin-dependent kinase 12 (CDK12) defines a metastatic castration-resistant prostate cancer (mCRPC) subtype. It remains unclear, however, whether CDK12 loss drives prostate cancer (PCa) development or uncovers pharmacologic vulnerabilities. Here, we show Cdk12 ablation in murine prostate epithelium is sufficient to induce preneoplastic lesions with lymphocytic infiltration. In allograft-based CRISPR screening, Cdk12 loss associates positively with Trp53 inactivation but negatively with Pten inactivation. Moreover, concurrent Cdk12/Trp53 ablation promotes proliferation of prostate-derived organoids, while Cdk12 knockout in Pten-null mice abrogates prostate tumor growth. In syngeneic systems, Cdk12/Trp53-null allografts exhibit luminal morphology and immune checkpoint blockade sensitivity. Mechanistically, Cdk12 inactivation mediates genomic instability by inducing transcription-replication conflicts. Strikingly, CDK12-mutant organoids and patient-derived xenografts are sensitive to inhibition or degradation of the paralog kinase, CDK13. We therein establish CDK12 as a bona fide tumor suppressor, mechanistically define how CDK12 inactivation causes genomic instability, and advance a therapeutic strategy for CDK12-mutant mCRPC.

α-l-(3-2)-Threofuranosyl nucleic acid (TNA) pairs with itself, cross-pairs with DNA and RNA, and shows promise as a tool in synthetic genetics, diagnostics, and oligonucleotide therapeutics. We studied in vitro primer insertion and extension reactions catalyzed by human trans-lesion synthesis (TLS) DNA polymerase η (hPol η) opposite a TNA-modified template strand without and in combination with O4-alkyl thymine lesions. Across TNA-T (tT), hPol η inserted mostly dAMP and dGMP, dTMP and dCMP with lower efficiencies, followed by extension of the primer to a full-length product. hPol η inserted dAMP opposite O4-methyl and -ethyl analogs of tT, albeit with reduced efficiencies relative to tT. Crystal structures of ternary hPol η complexes with template tT and O4-methyl tT at the insertion and extension stages demonstrated that the shorter backbone and different connectivity of TNA compared to DNA (3 → 2 versus 5 → 3, respectively) result in local differences in sugar orientations, adjacent phosphate spacings, and directions of glycosidic bonds. The 3-OH of the primers terminal thymine was positioned at 3.4 Å on average from the α-phosphate of the incoming dNTP, consistent with insertion opposite and extension past the TNA residue by hPol η. Conversely, the crystal structure of a ternary hPol η·DNA·tTTP complex revealed that the primers terminal 3-OH was too distant from the tTTP α-phosphate, consistent with the inability of the polymerase to incorporate TNA. Overall, our study provides a better understanding of the tolerance of a TLS DNA polymerase vis-à-vis unnatural nucleotides in the template and as the incoming nucleoside triphosphate.

Impaired control of the G1/S checkpoint allows initiation of DNA replication under non-permissive conditions. Unscheduled S-phase entry is associated with DNA replication stress, demanding for other checkpoints or cellular pathways to maintain proliferation. Here, we uncovered a requirement for ADARp150 to sustain proliferation of G1/S-checkpoint-defective cells under growth-restricting conditions. Besides its well-established mRNA editing function in inversely oriented short interspersed nuclear elements (SINEs), we found ADARp150 to exert a critical function in mitosis. ADARp150 depletion resulted in tetraploidization, impeding cell proliferation in mitogen-deprived conditions. Mechanistically we show that ADAR1 depletion induced aberrant expression of Cyclin B3, which was causative for mitotic failure and whole-genome duplication. Finally, we find that also in vivo ADAR1-depletion-provoked tetraploidization hampers tumor outgrowth.

In our recent publication, we have proposed a revised base excision repair pathway in which DNA polymerase β (Polβ) catalyzes Schiff base formation prior to the gap-filling DNA synthesis followed by β-elimination. In addition, the polymerase activity of Polβ employs the "three-metal ion mechanism" instead of the long-standing "two-metal ion mechanism" to catalyze phosphodiester bond formation based on the fact derived from time-resolved x-ray crystallography that a third Mg2+ was captured in the polymerase active site after the chemical reaction was initiated. In this study, we develop the models of the uncross-linked and cross-linked Polβ complexes and investigate the "three-metal ion mechanism" vs the "two-metal ion mechanism" by using the quantum mechanics/molecular mechanics molecular dynamics simulations. Our results suggest that the presence of the third Mg2+ ion stabilizes the reaction-state structures, strengthens correct nucleotide binding, and accelerates phosphodiester bond formation. The improved understanding of Polβ's catalytic mechanism provides valuable insights into DNA replication and damage repair. [ABSTRACT FROM AUTHOR]

DNA replication stress (RS) is a widespread phenomenon in carcinogenesis, causing genomic instability and extensive chromatin alterations. DNA damage leads to activation of innate immune signaling, but little is known about transcriptional regulators mediating such signaling upon RS. Using a chemical screen, we identified protein arginine methyltransferase 5 (PRMT5) as a key mediator of RS-dependent induction of interferon-stimulated genes (ISGs). This response is also associated with reactivation of endogenous retroviruses (ERVs). Using quantitative mass spectrometry, we identify proteins with PRMT5-dependent symmetric dimethylarginine (SDMA) modification induced upon RS. Among these, we show that PRMT5 targets and modulates the activity of ZNF326, a zinc finger protein essential for ISG response. Our data demonstrate a role for PRMT5-mediated SDMA in the context of RS-induced transcriptional induction, affecting physiological homeostasis and cancer therapy.

Cellular signaling, crucial for biological processes like immune response and homeostasis, relies on specificity and fidelity in signal transduction to accurately respond to stimuli amidst biological noise. Kinetic proofreading (KPR) is a key mechanism enhancing signaling specificity through time-delayed steps, although its effectiveness is debated due to intrinsic noise potentially reducing signal fidelity. In this study, we reformulate the theory of kinetic proofreading (KPR) by convolving multiple intermediate states into a single state and then define an overall processing time required to traverse these states. This simplification allows us to succinctly describe kinetic proofreading in terms of a single waiting time parameter, facilitating a more direct evaluation and comparison of KPR performance across different biological contexts such as DNA replication and T cell receptor (TCR) signaling. We find that loss of fidelity for longer proofreading steps relies on the specific strategy of information extraction and show that in the first-passage time (FPT) discrimination strategy, longer proofreading steps can exponentially improve the accuracy of KPR at the cost of speed. Thus, KPR can still be an effective discrimination mechanism in the high noise regime. However, in a product concentration-based discrimination strategy, longer proofreading steps do not necessarily lead to an increase in performance. However, by introducing activation thresholds on product concentrations, can we decompose the product-based strategy into a series of FPT-based strategies to better resolve the subtleties of KPR-mediated product discrimination. Our findings underscore the importance of understanding KPR in the context of how information is extracted and processed in the cell.

Artificial biomolecular condensates are emerging as a versatile approach to organize molecular targets and reactions without the need for lipid membranes. Here we ask whether the temporal response of artificial condensates can be controlled via designed chemical reactions. We address this general question by considering a model problem in which a phase separating component participates in reactions that dynamically activate or deactivate its ability to self-attract. Through a theoretical model we illustrate the transient and equilibrium effects of reactions, linking condensate response and reaction parameters. We experimentally realize our model problem using star-shaped DNA motifs known as nanostars to generate condensates, and we take advantage of strand invasion and displacement reactions to kinetically control the capacity of nanostars to interact. We demonstrate reversible dissolution and growth of DNA condensates in the presence of specific DNA inputs, and we characterize the role of toehold domains, nanostar size, and nanostar valency. Our results will support the development of artificial biomolecular condensates that can adapt to environmental changes with prescribed temporal dynamics.

Chromatin-based epigenetic memory relies on the accurate distribution of parental histone H3-H4 tetramers to newly replicated DNA strands. Mcm2, a subunit of the replicative helicase, and Dpb3/4, subunits of DNA polymerase ε, govern parental histone H3-H4 deposition to the lagging and leading strands, respectively. However, their contribution to epigenetic inheritance remains controversial. Here, using fission yeast heterochromatin inheritance systems that eliminate interference from initiation pathways, we show that a Mcm2 histone binding mutation severely disrupts heterochromatin inheritance, while mutations in Dpb3/4 cause only moderate defects. Surprisingly, simultaneous mutations of Mcm2 and Dpb3/4 stabilize heterochromatin inheritance. eSPAN (enrichment and sequencing of protein-associated nascent DNA) analyses confirmed the conservation of Mcm2 and Dpb3/4 functions in parental histone H3-H4 segregation, with their combined absence showing a more symmetric distribution of parental histone H3-H4 than either single mutation alone. Furthermore, the FACT histone chaperone regulates parental histone transfer to both strands and collaborates with Mcm2 and Dpb3/4 to maintain parental histone H3-H4 density and faithful heterochromatin inheritance. These results underscore the importance of both symmetric distribution of parental histones and their density at daughter strands for epigenetic inheritance and unveil distinctive properties of parental histone chaperones during DNA replication.

HBO1, also known as KAT7 or MYST2, is a crucial histone acetyltransferase with diverse cellular functions. It typically forms complexes with protein subunits or cofactors such as MEAF6, ING4, or ING5, and JADE1/2/3 or BRPF1/2/3, where the BRPF or JADE proteins serve as the scaffold targeting histone H3 or H4, respectively. The histone acetylation mediated by HBO1 plays significant roles in DNA replication and gene expression regulation. Additionally, HBO1 catalyzes the modification of proteins through acylation with propionyl, butyryl, crotonyl, benzoyl, and acetoacetyl groups. HBO1 undergoes ubiquitination and degradation by two types of ubiquitin complexes and can also act as an E3 ubiquitin ligase for the estrogen receptor α (ERα). Moreover, HBO1 participates in the expansion of medullary thymic epithelial cells (mTECs) and regulates the expression of peripheral tissue genes (PTGs) mediated by autoimmune regulator (AIRE), thus inducing immune tolerance. Furthermore, HBO1 influences the renewal of hematopoietic stem cells and the development of neural stem cells significantly. Importantly, the overexpression of HBO1 in various cancers suggests its carcinogenic role and potential as a therapeutic target. This review summarizes recent advancements in understanding HBO1's involvement in acylation modification, DNA replication, ubiquitination, immunity, and stem cell renewal. [ABSTRACT FROM AUTHOR]

Background: Staphylococcus aureus (S. aureus) mastitis results in economic losses during dairy production. Understanding the biological progression of bovine S. aureus mastitis is vital for its prevention. Lipoteichoic acid is a key virulence factor of S. aureus (aLTA), but the main biological pathways involved in its effect on bovine mammary epithetionallial cells (Mac-T) apoptosis and necrosis have not been fully explored. Folic acid (FA) has anti-inflammatory and anti-apoptotic effects. However, the role of FA in mediating the effects of aLTA on apoptosis and necrosis remains unknown. Results: We found that low concentration of aLTA inhibited apoptosis and necrosis and that high concentration promoted the apoptosis and necrosis of Mac-T. FA pretreatment alleviated high concentration of aLTA induced apoptosis. Through transcriptomic analysis, we found that nuclear receptor subfamily 4 group A (NR4A), which alters the expression of downstream genes involved in apoptosis, proliferation, and inflammation, decreased under stimulation with a low concentration of aLTA and increased under stimulation with a high concentration of aLTA. Under stimulation with a high concentration of aLTA, the expression of the NR4A subfamily could be inhibited by FA. The results showed that aLTA may affect apoptosis and necrosis through the NR4A subfamily by targeting genes involved in bacterial invasion of epithelial cells, the IL-17 signaling pathway, DNA replication, longevity regulation, the cell cycle, and tight junction pathways. We further found that the expression trends of NR4A1 and the target genes of the NR4A subfamily (PTGS2, ESPL1, MCM5, and BUB1B) in the blood of healthy cows (Healthy), subclinical mastitis cows (SCM), and SCM supplemented with FA (SCM_FA) were consistent with those observed at the cellular level in this study. Conclusions: Our study revealed that low and high concentrations of aLTA have opposite effects on apoptosis and necrosis of Mac-T and that FA can alleviate the apoptosis induced by high concentration of aLTA. Transcriptome analysis revealed that the NR4A subfamily play a role in the ability of FA to alleviate the apoptosis and necrosis induced by high concentration of aLTA. [ABSTRACT FROM AUTHOR]

Isothermal PCR can be performed by imposing a static temperature gradient that continuously circulates reagents through denaturing, annealing, and extension conditions inside a PCR tube. But despite early promise, these systems have yet to demonstrate performance and repeatability sufficient for adoption in validated laboratory tests because the rate‐limiting extension step is inherently short and cannot be increased independently of the other stages in a temperature cycle. Here, a discovery that enables isothermal PCR to be achieved with statistically robust repeatability that meets or exceeds diagnostic assay requirements (false positive/negative rate <8% at 95% confidence) by manipulating the interplay between the DNA replication biochemistry (via the amplicon GC content) and the microscale circulatory flow inside a PCR tube is reported. Surprisingly, optimal performance depends on selecting primer sequences that replicate high GC content amplicons, contradicting established PCR primer design rules. This innovative thermocycling approach accelerates PCR to speeds rivaling ultra‐fast instruments, enabling rapid, repeatable isothermal DNA analysis across a range of targets relevant to diagnostics and pathogen detection. [ABSTRACT FROM AUTHOR]

Starvation of Escherichia coli thyA auxotrophs for the required thymine or thymidine leads to the cessation of DNA synthesis and, unexpectedly, to thymineless death (TLD). Previously, TLD-alleviating defects were identified by the candidate gene approach, for their contribution to replication initiation, fork repair, or SOS induction. However, no TLD-blocking mutations were ever found, suggesting a multifactorial nature of TLD. Since (until recently) no unbiased isolation of TLD suppressors was reported, we used enrichment after insertional mutagenesis to systematically isolate TLD suppressors. Our approach was validated by isolation of known TLD-alleviating mutants in recombinational repair. At the same time, and unexpectedly for the current TLD models, most of the isolated suppressors affected general metabolism, while the strongest suppressors impacted the central metabolism. Several temperature-sensitive (Ts) mutants in important/essential functions, like nadA , ribB , or coa A, almost completely suppressed TLD at 42°C. Since blocking protein synthesis completely by chloramphenicol prevents TLD, while reducing protein synthesis to 10% alleviates TLD only slightly, we measured the level of protein synthesis in these mutants at 42°C and found it to be 20–70% of the WT, not enough reduction to explain TLD prevention. We conclude that the isolated central metabolism mutants prevent TLD by affecting specific TLD-promoting functions. [ABSTRACT FROM AUTHOR]

RNA–DNA hybrid is a part of the R‐loop which is an important non‐standard nucleic acid structure. RNA–DNA hybrid/R‐loop causes genomic instability by inducing DNA damages or inhibiting DNA replication. It also plays biologically important roles in regulation of transcription, replication, recombination and repair. Here, we have employed catalytically inactive human RNase H1 mutant (D145N) to visualize RNA–DNA hybrids and map their genomic locations in fission yeast cells. The RNA–DNA hybrids appear as multiple nuclear foci in rnh1∆rnh201∆ cells lacking cellular RNase H activity, but not in the wild‐type. The majority of RNA–DNA hybrid loci are detected at the protein coding regions and tRNA. In rnh1∆rnh201∆ cells, cells with multiple Rad52 foci increase during S‐phase and about 20% of the RNA–DNA hybrids overlap with Rad52 loci. During S‐phase, more robust association of Rad52 with RNA–DNA hybrids was observed in the protein coding region than in M‐phase. These results suggest that persistent RNA–DNA hybrids in the protein coding region in rnh1∆rnh201∆ cells generate DNA damages during S‐phase, potentially through collision with DNA replication forks. [ABSTRACT FROM AUTHOR]

Ganciclovir (GCV) is a clinically important drug as it is used to treat viral infections. GCV is incorporated into the DNA during replication, where it interferes with subsequent replication on GCV‐incorporated templates. However, the effects of GCV on the host genome and the mechanisms underlying cellular tolerance to GCV remain unclear. In this study, we explored these mechanisms using a collection of mutant DT40 cells. We identified RAD17/−, BRCA1−/−, and RAD18−/− cells as highly GCV‐sensitive. RAD17, a component of the alternative checkpoint‐clamp loader RAD17‐RFC, was required for the activation of the intra‐S checkpoint following GCV treatment. BRCA1, a critical factor for promoting homologous recombination (HR), was required for suppressing DNA double‐strand breaks (DSBs). Moreover, RAD18, an E3‐ligase involved in DNA repair, was critical in suppressing the aberrant ligation of broken chromosomes caused by GCV. We found that BRCA1 suppresses DSBs through HR‐mediated repair and template switching (TS)‐mediated damage bypass. Moreover, the strong GCV sensitivity of BRCA1−/− cells was rescued by the loss of 53BP1, despite the only partial restoration in the sister chromatid exchange events which are hallmarks of HR. These results indicate that BRCA1 promotes cellular tolerance to GCV through two mechanisms, TS and HR‐mediated repair. [ABSTRACT FROM AUTHOR]

Proliferating cell nuclear antigen (PCNA) plays a critical role in DNA replication by enhancing the activity of various proteins involved in replication. In this study, the crystal structure of ApePCNA1, one of three PCNAs from the thermophilic archaeon Aeropyrum pernix, was elucidated. ApePCNA1 was cloned and expressed in Escherichia coli and the protein was purified and crystallized. The resulting crystal structure determined at 2.00 Å resolution revealed that ApePCNA1 does not form a trimeric ring, unlike PCNAs from other domains of life. It has unique structural features, including a long interdomain‐connecting loop and a PIP‐box‐like sequence at the N‐terminus, indicating potential interactions with other proteins. These findings provide insights into the functional mechanisms of PCNAs in archaea and their evolutionary conservation across different domains of life. A modified medium and protocol were used to express recombinant protein containing the lac operon. The expression of the target protein increased and the total incubation time decreased when using this system compared with those of previous expression protocols. [ABSTRACT FROM AUTHOR]

Methicillin-Resistant Staphylococcus aureus forming into biofilms can trigger chronic inflammation and disrupt skin wound healing processes. Prolonged and excessive use of antibiotics can expedite the development of resistance, primarily because of their limited ability to penetrate microbial membranes and biofilms, especially antibiotics with intracellular drug targets. Herein, we devise a strategy in which virus-inspired nanoparticles control the release of antibiotics through rapid penetration into both bacterial cells and biofilms, thereby combating antimicrobial-resistant infections and promoting skin wound healing. Lipid-based nanoparticles based on stearamine and cholesterol were designed to mimic viral highly ordered nanostructures. To mimic the arginine-rich fragments in viral protein transduction domains, the primary amines on the surface of the lipid-based nanoparticles were exchanged by guanidine segments. Levofloxacin, an antibiotic that inhibits DNA replication, was chosen as the model drug to be incorporated into nanoparticles. Hyaluronic acid was coated on the surface of nanoparticles acting as a capping agent to achieve bacterial-specific degradation and guanidine explosion in the bacterial microenvironment. Our virus-inspired nanoparticles displayed long-acting antibacterial effects and powerful biofilm elimination to overcome antimicrobial-resistant infections and promote skin wound healing. This work demonstrates the ability of virus-inspired nanoparticles to achieve a dual penetration of microbial cell membranes and biofilm structures to address antimicrobial-resistant infections and trigger skin wound healing. [Display omitted] • This work demonstrates virus-inspired nanoparticles for solving bacterial infection. • Nanoparticles deeply penetrate microbial membranes and biofilm structures. • Nanoparticles exhibit long-term antimicrobial activity with low resistance risk. • Nanoparticles defeat microbial through a multitarget interference approach. • Nanoparticles accelerate skin wound repair and display excellent biosafety. [ABSTRACT FROM AUTHOR]

Our previous study has demonstrated that the decline in cardiomyocytes proliferation capacity induced by maternal androgen excess was mainly attributed to the accumulation of androsterone in the heart. However, the underlying mechanism by which androsterone inhibits cardiomyocytes proliferation remains unknown. In this study, pregnant mice were injected subcutaneously daily with dihydrotestosterone (DHT) from gestational day (GD) 16.5 to GD18.5. On GD18.5, fetal heart tissue was dissected and used for analyzing androgen receptor (AR) levels. H9c2 cells and primary cardiomyocytes, isolated from fetal hearts, were applied to investigate the mechanism. H9c2 cells under androsterone treatment were subjected to RNA sequencing analysis and the results showed that genes were primarily enriched in cell cycle and DNA replication pathways. Elevated AR levels were observed in fetal cardiac tissue in the maternal DHT‐treated group. Androsterone treatment increased the ratio of nuclear AR and cytoplasmic AR both in H9c2 cells and primary cardiomyocytes. The ablation and overexpression of AR can mildly reverse and aggravate cell cycle arrest induced by androsterone, respectively. ChIP‐qPCR analysis suggested that AR can directly repress cell cycle and DNA replication‐related gene expression, which was mediated by the recruitment of retinoblastoma protein (Rb). The repression of cell proliferation in response to androsterone was alleviated partly through the downregulation of Rb by siRNA transfection. In conclusion, AR repression to cell cycle and DNA replication‐related gene expression, mediated by recruitment of Rb, may be one of the potential mechanisms of cell cycle arrest in cardiomyocytes induced by androsterone. [ABSTRACT FROM AUTHOR]

In chromatin replication, faithful recycling of histones from parental DNA to replicated strands is essential for maintaining epigenetic information across generations. A previous experiment has revealed that disrupting interactions between the N-terminal tail of Mcm2, a subunit in DNA replication machinery, and a histone H3/H4 tetramer perturb the recycling. However, the molecular pathways and the factors that regulate the ratio recycled to each strand and the destination location are yet to be revealed. Here, we performed molecular dynamics simulations of yeast DNA replication machinery, an H3/H4 tetramer, and replicated DNA strands. The simulations demonstrated that histones are recycled via Cdc45-mediated and unmediated pathways without histone chaperones, as our in vitro biochemical assays supported. Also, RPA binding regulated the ratio recycled to each strand, whereas DNA bending by Pol ε modulated the destination location. Together, the simulations provided testable hypotheses, which are vital for elucidating the molecular mechanisms of histone recycling. Parental histones are faithfully recycled in chromatin replication. Here the authors performed molecular dynamics simulations of replication machinery and proposed that histones are recycled via Cdc45-mediated and unmediated pathways. [ABSTRACT FROM AUTHOR]

Cardiovascular disease is the deadly disease that can result in sudden death, and inflammation plays an important role in its onset and progression. High mobility group box 1 (HMGB1) is a nuclear protein that regulates transcription, DNA replication, repair, and nucleosome assembly. HMGB1 is released passively by necrotic tissues and actively secreted by stressed cells. Extracellular HMGB1 functions as a damage associated molecular patterns molecule, producing numerous redox forms that induce a range of cellular responses by binding to distinct receptors and interactors, including tissue inflammation and regeneration. Extracellular HMGB1 inhibition reduces inflammation and is protective in experimental models of myocardial ischemia/reperfusion damage, myocarditis, cardiomyopathies caused by mechanical stress, diabetes, bacterial infection, or chemotherapeutic drugs. HMGB1 administration following a myocardial infarction followed by permanent coronary artery ligation improves cardiac function by stimulating tissue regeneration. HMGB1 inhibits contractility and produces hypertrophy and death in cardiomyocytes, while also stimulating cardiac fibroblast activity and promoting cardiac stem cell proliferation and differentiation. Maintaining normal nuclear HMGB1 levels, interestingly, protects cardiomyocytes from apoptosis by limiting DNA oxidative stress, and mice with HMGB1cardiomyocyte-specific overexpression are partially protected from cardiac injury. Finally, elevated levels of circulating HMGB1 have been linked to human heart disease. As a result, following cardiac damage, HMGB1 elicits both detrimental and helpful responses, which may be due to the formation and stability of the various redox forms, the particular activities of which in this context are mostly unknown. This review covers recent findings in HMGB1 biology and cardiac dysfunction. [ABSTRACT FROM AUTHOR]

The genome duplication program is affected by multiple factors in vivo , including developmental cues, genotoxic stress, and aging. Here, we monitored DNA replication initiation dynamics in regenerating livers of young and old mice after partial hepatectomy to investigate the impact of aging. In young mice, the origin firing sites were well defined; the majority were located 10–50 kb upstream or downstream of expressed genes, and their position on the genome was conserved in human cells. Old mice displayed the same replication initiation sites, but origin firing was inefficient and accompanied by a replication stress response. Inhibitors of the ATR checkpoint kinase fully restored origin firing efficiency in the old mice but at the expense of an inflammatory response and without significantly enhancing the fraction of hepatocytes entering the cell cycle. These findings unveil aging-dependent replication stress and a crucial role of ATR in mitigating the stress-associated inflammation, a hallmark of aging. [Display omitted] • Partial hepatectomy enables in vivo mapping of DNA replication origins in mouse livers • Most origins are located 10–50 kb upstream or downstream of expressed genes • An aging-dependent reduction in origin firing is mediated by the ATR checkpoint • ATR inhibitors restore origin firing in old mice but lead to liver inflammation In vivo DNA replication dynamics in regenerating mouse livers unveil aging-dependent decline in replication efficiency and a crucial role of the ATR checkpoint kinase in mitigating inflammation associated with the decline. [ABSTRACT FROM AUTHOR]

Telomeres are the protective caps at the ends of linear chromosomes of eukaryotic organisms. Telomere binding proteins, including the six components of the complex known as shelterin, mediate the protective function of telomeres. They do this by suppressing many arms of the canonical DNA damage response, thereby preventing inappropriate fusion, resection and recombination of telomeres. One way this is achieved is by facilitation of DNA replication through telomeres, thus protecting against a "replication stress" response and activation of the master kinase ATR. On the other hand, DNA damage responses, including replication stress and ATR, serve a positive role at telomeres, acting as a trigger for recruitment of the telomere-elongating enzyme telomerase to counteract telomere loss. We postulate that repression of telomeric replication stress is a shared mechanism of control of telomerase recruitment and telomere length, common to several core telomere binding proteins including TRF1, POT1 and CTC1. The mechanisms by which replication stress and ATR cause recruitment of telomerase are not fully elucidated, but involve formation of nuclear actin filaments that serve as anchors for stressed telomeres. Perturbed control of telomeric replication stress by mutations in core telomere binding proteins can therefore cause the deregulation of telomere length control characteristic of diseases such as cancer and telomere biology disorders. [ABSTRACT FROM AUTHOR]

Background: Synpolydactyly (SPD) is mainly caused by mutations of polyalanine expansion (PAE) in the transcription factor gene HOXD13 and the involved cell types and signal pathway are still not clear possible pathways and single-cell expression characteristics of limb bud in HOXD13 PAE mice was analyzed in this study. Method: We investigated a previous study of a mouse model with SPD and conducted weighted gene co-expression network analysis (WGCNA) using a single-cell RNA sequencing dataset from limb bud cells of SPD mouse model of HOXD13 + 7A heterozygote. Results: Analysis of WGCNA revealed that synpolydactyly-associated Hoxd13 PAEs alter the immune response and osteoclast differentiation, and enhance DNA replication. Bmp4, Hand2, Hoxd12, Lnp, Prrx1, Gmnn, and Cdc6 were found to play potentially key roles in synpolydactyly. Conclusions: These findings evaluated the main genes related to SPD with PAE mutations in HOXD13 and advance our understanding of human limb development. [ABSTRACT FROM AUTHOR]

Breast cancer is associated with high morbidity and mortality, which are closely influenced by protein post‐translational modifications (PTMs). Lysine crotonylation (Kcr) serves as a newly identified PTM type that plays a role in various biological processes; however, its involvement in breast cancer progression remains unclear. Minichromosome maintenance 6 (MCM6) is a critical component of DNA replication and has been previous confirmed to exhibit a significant role in tumorigenesis. Despite this, a comprehensive analysis of MCM6, particularly regarding its modifications in breast cancer is lacking. In this study, we found MCM6 is upregulated in breast invasive carcinoma (BRCA) and is associated with poorer overall survival by regulating the DNA damage repair mechanisms. Furthermore, MCM6‐knockdown resulted in decreased cell proliferation and inhibited the DNA replication, leading to DNA replication stress and sustained DNA damage, thereby enhancing the chemotherapeutic sensitivity of breast cancer. Additionally, SIRT7‐mediated crotonylation of MCM6 at K599 (MCM6‐K599cr) was significantly upregulated in response to DNA replication stress, primarily due to the disassemebly of the MCM2‐7 complex and regulated by RNF8‐mediated ubiquitination. Concurrently, kaempferol, which acts as a regulator of SIRT7, was found to enhance the Kcr level of MCM6, reducing tumour weight, particular when combined with paclitaxel, highlighting its potential chemotherapeutic target for BRCA therapy. [ABSTRACT FROM AUTHOR]

In eukaryotes, maintenance of heterochromatin structure that represses gene expression during cell proliferation is essential for guaranteeing cell identity. However, how heterochromatin is maintained and transmitted to the daughter cells remains elusive. In this study, we constructed a reporter system to study the maintenance of heterochromatin in the subtelomeric region of the fission yeast, Schizosaccharomyces pombe. We demonstrated that once subtelomeric heterochromatin was established, it tended to be maintained as a metastable structure through cell proliferation. Using this system, we screened an S. pombe genome‐wide gene deletion library for subtelomeric heterochromatin maintenance factors and identified 57 genes related to various cellular processes, in addition to well‐characterized heterochromatin factors. We focused on Mrc1Claspin, a mediator of DNA replication checkpoint. We found that Mrc1 maintains heterochromatin structure not only at the subtelomeres but also at the pericentromeres and mating‐type regions. Furthermore, we showed that Mrc1 is required for the localization of Snf2/Hdac‐containing Repressor Complex (SHREC) and the maintenance of hypoacetylation state of histone H3K14. This study complements the recent discoveries that Mrc1 functions as a histone H3‐H4 chaperone in heterochromatin maintenance. [ABSTRACT FROM AUTHOR]

The Mpox virus (or Monkeypox virus, MPXV) uses its own encoded proteins to form a replication machine that replicates the viral genome in the host cell cytoplasm, making this machinery a key target for antiviral drug design. The D5 (also known as the OPG117 or E5) protein, a bi‐functional helicase–primase enzyme, is crucial in the MPXV replication machinery and genome uncoating process. Recently, cryo‐electron microscopy (cryo‐EM) structures of D5 in multiple states have been determined. These structures have elucidated the full trajectory of the MPXV D5 helicase–primase as it moves along single‐stranded DNA, providing unprecedented advancements in the molecular dynamics and unwinding mechanism. This structural snapshot describes the structural features of the D5 protein and dissects the broader implications of its pivotal role in MPXV replication. [ABSTRACT FROM AUTHOR]

The pluripotent mouse embryonic stem cell (mESCs) can transit into the totipotent-like state, and the transcription factor DUX is one of the master regulators of this transition. Intriguingly, this transition in mESCs is accompanied by massive cell death, which significantly impedes the establishment and maintenance of totipotent cells in vitro, yet the underlying mechanisms of this cell death remain largely elusive. In this study, we found that the totipotency transition in mESCs triggered cell death through the upregulation of DUX. Specifically, R-loops are accumulated upon DUX induction, which subsequently lead to DNA replication stress (RS) in mESCs. This RS further activates p53 and PMAIP1, ultimately leading to Caspase-9/7-dependent intrinsic apoptosis. Notably, inhibiting this intrinsic apoptosis not only mitigates cell death but also enhances the efficiency of the totipotency transition in mESCs. Our findings thus elucidate one of the mechanisms underlying cell apoptosis during the totipotency transition in mESCs and provide a strategy for optimizing the establishment and maintenance of totipotent cells in vitro. [ABSTRACT FROM AUTHOR]

ABSTRACT A novel Schiff base ligand, ((4‐oxo‐4H‐chromen‐3‐yl)methylene)isonicotinohydrazone (HL), in addition to its VO(II), Cd(II), Mn(II), and Pd(II) complexes, were prepared and identified. Various analytical techniques such as elemental, TGA, and magnetic susceptibility measurements, additionally, nuclear magnetic resonance (NMR) spectroscopy of hydrogen and carbon‐13, MS, FT‐IR, EPR, and electronic (UV‐VIS) spectroscopy, are utilized and proposed that HL has a mono‐negative tridentate mode of chelation with VO(II) and Mn(II) complexes, whereas it displays a mono‐negative bidentate with the Cd(II) complex and a neutral bidentate with the Pd(II) chelate. The crystallographic structures and atomic spacing for both HL and its complexes were examined through powder X‐ray diffraction analysis. Moreover, electrochemical analyses were conducted on Cd(II) and Mn(II) metal ions with and without the agent that chelates for the purpose of foreseeing the consequences of complex interactions in the case of the liquid phase. Furthermore, photoluminescence study was applied on VO(II) and Pd(II) complexes and showed a quenching effect for both. The ligand's geometry and its complexes were examined through calculations based on density functional theory (DFT) using B3LYP/LANL2DZ computational method and showed molecular electrostatic potential, chemical reactivity, and FMOs. The Cd(II) complex showed promising results against various bacterial strains, displaying excellent activity that exceeded that of the standard, Gentamicin (108% against K. pneumoniae, 100% against Salmonella, and 90.47% against A. fungi). In contrast, it exhibited no cytotoxicity toward the MCF‐7 cell line. However, in a DNA binding study, the compound demonstrated significant activity as its concentration increased up to 6 mM, suggesting its potential for use in cancer therapy due to its ability to inhibit DNA replication. [ABSTRACT FROM AUTHOR]

SUMMARY Among flowering plants, genome size varies remarkably, by >2200‐fold, and this variation depends on the loss and gain of noncoding DNA sequences that form distinct heterochromatin complexes during interphase. In plants with giant genomes, most chromatin remains condensed during interphase, forming a dense network of heterochromatin threads called interphase chromonemata. Using super‐resolution light and electron microscopy, we studied the ultrastructure of chromonemata during and after replication in root meristem nuclei of Nigella damascena L. During S‐phase, heterochromatin undergoes transient decondensation locally at DNA replication sites. Due to the abundance of heterochromatin, the replication leads to a robust disassembly of the chromonema meshwork and a general reorganization of the nuclear morphology visible even by conventional light microscopy. After replication, heterochromatin recondenses, restoring the chromonema structure. Thus, we show that heterochromatin replication in interphase nuclei of giant‐genome plants induces a global nuclear reorganization. [ABSTRACT FROM AUTHOR]

The acquisition of mitochondria was imperative for initiating eukaryogenesis and thus is a characteristic feature of eukaryotic cells. 1,2 The parasitic protist Trypanosoma brucei contains a singular mitochondrion with a unique mitochondrial genome, termed the kinetoplast DNA (kDNA). 3 Replication of the kDNA occurs during the G 1 phase of the cell cycle, prior to the start of nuclear DNA replication. 4 Although numerous proteins have been functionally characterized and identified as vital components of kDNA replication and division, the molecular mechanisms governing this highly precise process remain largely unknown. 5,6 One division-related and morphologically characteristic structure that remains most enigmatic is the "nabelschnur," an undefined, filament-resembling structure observed by electron microscopy between segregating daughter kDNA networks. 7,8,9 To date, only one protein, TbLAP1, an M17 family leucyl aminopeptidase metalloprotease, is known to localize to the nabelschnur. 9 While screening proteins from the T. brucei MitoTag project, 10 we identified a previously uncharacterized protein with an mNeonGreen signal localizing to the kDNA as well as forming a point of connection between dividing kDNAs. Here, we demonstrate that this kDNA-associated protein, named TbNAB70, indeed localizes to the nabelschnur and plays an essential role in the segregation of newly replicated kDNAs and subsequent cytokinesis in T. brucei. [Display omitted] • TbNAB70 localizes to both the kDNA and the nabelschnur in Trypanosoma brucei • TbNAB70 is identified to be vital for the segregation, but not replication, of kDNA • TbNAB70 depletion results in a near-complete arrest of cytokinesis • TbNAB70 evolutionary emergence occurred after the development of the kDNA network Cadena et al. identify TbNAB70 as localizing to the "nabelschnur," an enigmatic structure observed between dividing kDNA networks in Trypanosoma brucei. TbNAB70 is shown to be essential for the segregation of kDNA and basal bodies, while the loss of TbNAB70 results in the arrest of cytokinesis, highlighting its critical role in cellular division processes. [ABSTRACT FROM AUTHOR]

DNA damage is a primary trigger for cellular senescence, which in turn causes organismal aging and is a promising target of anti-aging therapies. Most DNA damage occurs when DNA is fragile during DNA replication in S phase, but senescent cells maintain DNA damage long-after DNA replication has stopped. How senescent cells induce DNA damage and why senescent cells fail to repair damaged DNA remain open questions. Here, we combine reversible expression of the senescence-inducing CDK4/6 inhibitory protein p16INK4 (p16) with live single-cell analysis and show that sustained mTORC1 signaling triggers senescence in non-proliferating cells by increasing transcriptional DNA damage and inflammation signaling that persists after p16 is degraded. Strikingly, we show that activation of E2F transcriptional program, which is regulated by CDK4/6 activity and promotes expression of DNA repair proteins, repairs transcriptionally damaged DNA without requiring DNA replication. Together, our study suggests that senescence can be maintained by ongoing mTORC1-induced transcriptional DNA damage that cannot be sufficiently repaired without induction of protective E2F target genes. Persistent DNA damage is a hallmark of senescence. Here, the authors show that senescent cells accumulate DNA damage due to transcriptional stress and are unable to repair DNA damage due to the absence of cell-cycle regulated DNA repair programs. [ABSTRACT FROM AUTHOR]

Replication, heredity, and evolution are characteristic of Life. We and others have postulated that the reconstruction of a synthetic living system in the laboratory will be contingent on the development of a genetic self-replicator capable of undergoing Darwinian evolution. Although DNA-based life dominates, the in vitro reconstitution of an evolving DNA self-replicator has remained challenging. We hereby emulate in liposome compartments the principles according to which life propagates information and evolves. Using two different experimental configurations supporting intermittent or semi-continuous evolution (i.e., with or without DNA extraction, PCR, and re-encapsulation), we demonstrate sustainable replication of a linear DNA template – encoding the DNA polymerase and terminal protein from the Phi29 bacteriophage – expressed in the 'protein synthesis using recombinant elements' (PURE) system. The self-replicator can survive across multiple rounds of replication-coupled transcription-translation reactions in liposomes and, within only ten evolution rounds, accumulates mutations conferring a selection advantage. Combined data from next-generation sequencing with reverse engineering of some of the enriched mutations reveal nontrivial and context-dependent effects of the introduced mutations. The present results are foundational to build up genetic complexity in an evolving synthetic cell, as well as to study evolutionary processes in a minimal cell-free system. The construction of a synthetic cell will be contingent on the development of an evolvable genetic replicator. Here, authors create a Darwinian protocell that supports protein synthesis, DNA self-replication, and adaptive evolution. [ABSTRACT FROM AUTHOR]

R-loops are three-stranded nucleic acid structures composed of an RNA–DNA hybrid and a displaced DNA strand. They are widespread and play crucial roles in regulating gene expression, DNA replication, and DNA and histone modifications. However, their regulatory mechanisms remain unclear. As R-loop detection technology advances, changes in R-loop levels have been observed in cancer models, often associated with transcription-replication conflicts and genomic instability. N6-methyladenosine (m6A) is an RNA epigenetic modification that regulates gene expression by affecting RNA localization, splicing, translation, and degradation. Upon reviewing the literature, we found that R-loops with m6A modifications are implicated in tumor development and progression. This article summarizes the molecular mechanisms and detection methods of R-loops and m6A modifications in gene regulation, and reviews recent research on m6A-modified R-loops in oncology. Our goal is to provide new insights into the origins of genomic instability in cancer and potential strategies for targeted therapy. [ABSTRACT FROM AUTHOR]

The early stages of embryonic development rely on maternal products for proper regulation. However, screening for functional maternal-specific factors is challenging due to the time- and labor-intensive nature of traditional approaches. Here, we combine a computational pipeline and F0 null mutant technology to screen for functional maternal-specific chromatin regulators in zebrafish embryogenesis and identify Mcm3l, Mcm6l, and Npm2a as playing essential roles in DNA replication and cell division. Our results contribute to understanding the molecular mechanisms underlying early embryo development and highlight the importance of maternal-specific chromatin regulators in this critical stage. Computational tools and F0 null mutant analysis allow the identification of the maternal chromatin regulators Mcm3l, Mcm6l, and Npm2a in zebrafish embryogenesis, which are essential for DNA replication and cell division. [ABSTRACT FROM AUTHOR]

Chromothripsis describes the catastrophic shattering of mis-segregated chromosomes trapped within micronuclei. Although micronuclei accumulate DNA double-strand breaks and replication defects throughout interphase, how chromosomes undergo shattering remains unresolved. Using CRISPR-Cas9 screens, we identify a non-canonical role of the Fanconi anemia (FA) pathway as a driver of chromothripsis. Inactivation of the FA pathway suppresses chromosome shattering during mitosis without impacting interphase-associated defects within micronuclei. Mono-ubiquitination of FANCI-FANCD2 by the FA core complex promotes its mitotic engagement with under-replicated micronuclear chromosomes. The structure-selective SLX4-XPF-ERCC1 endonuclease subsequently induces large-scale nucleolytic cleavage of persistent DNA replication intermediates, which stimulates POLD3-dependent mitotic DNA synthesis to prime shattered fragments for reassembly in the ensuing cell cycle. Notably, FA-pathway-induced chromothripsis generates complex genomic rearrangements and extrachromosomal DNA that confer acquired resistance to anti-cancer therapies. Our findings demonstrate how pathological activation of a central DNA repair mechanism paradoxically triggers cancer genome evolution through chromothripsis. [Display omitted] • The FA pathway promotes the catastrophic shattering of micronuclear chromosomes • SLX4-XPF-ERCC1 cleaves mitotic DNA replication intermediates from micronuclei • MiDAS facilitates the mutagenic reassembly of shattered chromosomes in interphase • FA-induced mitotic shattering drives genome rearrangements and ecDNA formation A genetic screen uncovers the Fanconi anemia pathway as a driver of chromothripsis by shattering under-replicated micronuclear chromosomes during mitosis. This process involves chromosome-scale cleavage by the SLX4-XPF-ERCC1 endonuclease coupled to mitotic DNA synthesis, resulting in complex rearrangements and ecDNAs that fuel cancer genome evolution and/or resistance to anti-cancer therapies. [ABSTRACT FROM AUTHOR]

Simple Summary: When oncogenes, which are genes that can cause cancer, become active, they disrupt normal cell processes, especially DNA replication. This disruption is known as replication stress (RS), in which the DNA copying process is stalled or damaged. To prevent cancer, cells usually have a defense system called the DNA damage response (DDR), which can prevent damaged cells from turning into cancer. However, some cells manage to survive this stress by developing replication stress tolerance (RST). These cells can continue to grow, leading to genomic instability (GIN), which is a key feature of cancer. GIN causes various genetic changes that make cells more likely to become cancerous and more difficult to treat. This review explains how oncogenes cause RS and how cells cope with it, leading to the development of cancer. Understanding these processes can help in developing new cancer treatments. Activation of oncogenes disturbs a wide variety of cellular processes and induces physiological dysregulation of DNA replication, widely referred to as replication stress (RS). Oncogene-induced RS can cause replication forks to stall or collapse, thereby leading to DNA damage. While the DNA damage response (DDR) can provoke an anti-tumor barrier to prevent the development of cancer, a small subset of cells triggers replication stress tolerance (RST), allowing precancerous cells to survive, thereby promoting clonal expansion and genomic instability (GIN). Genomic instability (GIN) is a hallmark of cancer, driving genetic alterations ranging from nucleotide changes to aneuploidy. These alterations increase the probability of oncogenic events and create a heterogeneous cell population with an enhanced ability to evolve. This review explores how major oncogenes such as RAS, cyclin E, and MYC induce RS through diverse mechanisms. Additionally, we delve into the strategies employed by normal and cancer cells to tolerate RS and promote GIN. Understanding the intricate relationship between oncogene activation, RS, and GIN is crucial to better understand how cancer cells emerge and to develop potential cancer therapies that target these vulnerabilities. [ABSTRACT FROM AUTHOR]

DNA-RNA hybrid duplexes play essential roles during the reverse transcription of RNA viruses and DNA replication. The opening and conformation changes of individual base pairs are critical to their biological functions. However, the microscopic mechanisms governing base pair closing and opening at the atomic level remain poorly understood. In this study, we investigated the thermodynamic and kinetic parameters of the dA-rU base pair in a DNA-RNA hybrid duplex using 4 μs all-atom molecular dynamics (MD) simulations at different temperatures. Our results showed that the thermodynamic parameters of the dA-rU base pair aligned with the predictions of the nearest-neighbor model and were close to those of the AU base pair in RNA. The temperature dependence of the average lifetimes of both the open and the closed states, as well as the transition path times, were obtained. The free-energy barrier for a single base pair opening and closing arises from an increase in enthalpy due to the disruption of the base-stacking interactions and hydrogen bonding, along with an entropy loss attributed to the accompanying restrictions, such as torsional angle constraints and solvent viscosity. [ABSTRACT FROM AUTHOR]

Essential genes are crucial for microbial viability, playing key roles in both the primary and secondary metabolism. Since mutations in these genes can threaten organism viability, identifying them is challenging. Conditionally essential genes are required only under specific conditions and are important for functions such as virulence, immunity, stress survival, and antibiotic resistance. Transposon-directed sequencing (Tn-Seq) has emerged as a powerful method for identifying both essential and conditionally essential genes. In this review, we explored Tn-Seq workflows, focusing on eubacterial species and some yeast species. A comparison of 14 eubacteria species revealed 133 conserved essential genes, including those involved in cell division (e.g., ftsA, ftsZ), DNA replication (e.g., dnaA, dnaE), ribosomal function, cell wall synthesis (e.g., murB, murC), and amino acid synthesis (e.g., alaS, argS). Many other essential genes lack clear orthologues across different microorganisms, making them specific to each organism studied. Conditionally essential genes were identified in 18 bacterial species grown under various conditions, but their conservation was low, reflecting dependence on specific environments and microorganisms. Advances in Tn-Seq are expected to reveal more essential genes in the near future, deepening our understanding of microbial biology and enhancing our ability to manipulate microbial growth, as well as both the primary and secondary metabolism. [ABSTRACT FROM AUTHOR]

Microorganisms that exist in the seawater form microbial biofilms on materials used in marine construction, especially on metal surfaces submerged in seawater, where they form biofilms and cause severe corrosion. Biofilms are mainly composed of bacteria and their secreted polymeric substances. In order to understand how biofilms promote metal corrosion, planktonic and biofilm cells of Desulfovibrio bizertensis SY-1 (D. bizertensis) from Q235 steel were collected and analyzed as to their intracellular proteome and extracellular polymeric substances (EPS). The intracellular proteome analysis showed that the cellular proteins were strongly regulated in biofilm cells compared to planktonic cells, e.g., along with flagellar proteins, signaling-related proteins were significantly increased, whereas energy production and conversion proteins and DNA replication proteins were significantly regulated. The up-and-down regulation of proteins revealed that biofilm formation by bacteria on metal surfaces is affected by flagellar and signaling proteins. A significant decrease in DNA replication proteins indicated that DNA is no longer replicated and transcribed in mature biofilms, thus reducing energy consumption. Quantitative analysis and lectin staining of the biofilm on the metal's surface revealed that the bacteria secreted a substantial amount of EPS when they began to attach to the surface, and proteins dominated the main components of EPS. Further, the infrared analysis showed that the secondary structure of the proteins in the EPS of the biofilm was mainly dominated by β-sheet and 3-turn helix, which may help to enhance the adhesion of EPS. The functional groups of EPS analyzed using XPS showed that the C element of EPS in the biofilm mainly existed in the form of combinations with N. Furthermore, the hydroxyl structure in the EPS extracted from the biofilm had a stronger hydrogen bonding effect, which could maintain the stability of the EPS structure and biofilm. The study results revealed that D. bizertensis regulates the metabolic pathways and their secreted EPS structure to affect biofilm formation and cause metal corrosion, which has a certain reference significance for the study of the microbially influenced corrosion (MIC) mechanism. [ABSTRACT FROM AUTHOR]

Deoxyribonucleic acid (DNA) is able to form non-canonical four-stranded helical structures with diverse folding patterns known as G-quadruplexes (G4s). G4 topologies are classified based on their relative strand orientation following the 5' to 3' phosphate backbone polarity. Broadly, G4 topologies are either parallel (4+0), antiparallel (2+2), or hybrid (3+1). G4s play crucial roles in biological processes such as DNA repair, DNA replication, transcription and have thus emerged as biological targets in drug design. While computational models have been developed to predict G4 formation, there is currently no existing model capable of predicting G4 folding topology based on its nucleic acid sequence. Therefore, we introduce G4ShapePredictor (G4SP), an application featuring a collection of multi-classification machine learning models that are trained on a custom G4 dataset combining entries from existing literature and in-house circular dichroism experiments. G4ShapePredictor is designed to accurately predict G4 folding topologies in potassium ( K + ) buffer based on its primary sequence and is able to incorporate a threshold optimization strategy allowing users to maximise precision. Furthermore, we have identified three topological sequence motifs that suggest specific G4 folding topologies of (4+0), (2+2) or (3+1) when utilising the decision-making mechanisms of G4ShapePredictor. [ABSTRACT FROM AUTHOR]

Cervical cancer begins in the cells lining the cervix and is caused by persistent infection with certain types of human papillomavirus (HPV). Initially, it has no symptoms, and later it causes pelvic pain, abnormal vaginal bleeding, and pain during intercourse. It is the fourth-ranked cancer among women, and many women die from cervical cancer every year, particularly in low-income countries and the majority could be prevented with early detection and treatment. In this study, we have taken Cervical Cancer DNA Replication and Repair-Related Protein with the PDBID- 3H15, 5VBN, and 6NT9 and performed the multitargeted molecular docking with the FDA-approved drug library using HTVS, SP and XP docking. Then, the poses were filtered with MM\GBSA for proper computations of free energy, identified a multitargeted inhibitor Droxidopa with docking and MM\GBSA scores ranging from − 5.559 to − 6.835 Kcal/mol and − 26.04 to − 37.33 Kcal/mol, respectively. We also performed interaction fingerprints revealing 2VAL, 2LYS, 1ALA, 1ARG, 1ASN, 1CYS, 1GLN, 1GLU, 1ILE, 1MET, 1PHE, 1PRO, 1SER, and 1THR were most interacted residues and computed the ADMET properties with QikProp and DFT with Jaguar, which supported the study and compounds' suitability. Moreover, we performed the 100ns MD simulation in water, showing the controlled deviation and fluctuations of the residues with many interactions, and MM\GBSA was performed with the same trajectories, showing a better understanding of each frame's total complex and binding-free energy. The whole study favours droxidopa as an inhibitor of cervical cancer DNA Replication and Repair-Related Proteins—however, experimental studies are needed before use. [ABSTRACT FROM AUTHOR]

Branched DNA molecules are key intermediates in the molecular pathways of DNA replication, repair and recombination. Understanding their structural details, therefore, helps to envisage the mechanisms underlying these processes. While the configurations of DNA molecules can be effectively analysed in bulk using gel electrophoresis techniques, direct visualization provides a complementary single-molecule approach to investigating branched DNA structures. However, for microscopic examination, the sample needs to be free from impurities that could obscure the molecules of interest, and free from the bulk of unwanted non-specific DNA molecules that would otherwise dominate the field of view. Additionally, in the case of recombination intermediates, the length of the DNA molecules becomes an important factor to consider since the structures can be spread over a large distance on the chromosome in vivo. As a result, apart from sample purity, efficient isolation of large-sized DNA fragments without damaging their branched structures is crucial for further analysis. These factors are illustrated by the example of DNA double-strand break repair in the bacterium E. coli. In E. coli recombination intermediates may be spread over a distance of 40 kb which constitutes less than 1% of the 4.6 Mb genome. This study reveals ways to overcome some of the technical challenges that are associated with the isolation and purification of large and complex branched DNA structures using E. coli DNA double-strand break repair intermediates. High-molecular weight and branched DNA molecules do not run into agarose gels subjected to electrophoresis. However, they can be extracted from the wells of the gels if they are agarose embedded, by using β-agarase digestion, filtration, and concentration. Furthermore, a second round of gel electrophoresis followed by purification is recommended to enhance the purity of the specific DNA samples. These preliminary findings may prove to be pioneering for various single-molecule analyses of large and complex DNA molecules of DNA replication, repair and recombination. [ABSTRACT FROM AUTHOR]

Chronic tuberculosis (TB) disease, which requires months-long chemotherapy with multiple antibiotics, is defined by diverse pathological manifestations and bacterial phenotypes. Targeting drug-tolerant bacteria in the host is critical to achieving a faster and durable cure for TB. In order to facilitate this field of research, we need to consider the physiology of persistent MTB during infection, which is often associated with the nonreplicating (NR) state. However, the traditional approach to quantifying bacterial burden through colony enumeration alone only informs on the abundance of live bacilli at the time of sampling, and provides an incomplete picture of the replicative state of the pathogen and the extent to which bacterial replication is balanced by ongoing cell death. Modern approaches to profiling bacterial replication status provide a better understanding of inter- and intra-population dynamics under different culture conditions and in distinct host microenvironments. While some methods use molecular markers of DNA replication and cell division, other approaches take advantage of advances in the field of microfluidics and live-cell microscopy. Considerable effort has been made over the past few decades to develop preclinical in vivo models of TB infection and some are recognized for more closely recapitulating clinical disease pathology than others. Unique lesion compartments presenting different environmental conditions produce significant heterogeneity between Mycobacterium tuberculosis populations within the host. While cellular lesion compartments appear to be more permissive of ongoing bacterial replication, caseous foci are associated with the maintenance of M. tuberculosis in a state of static equilibrium. The accurate identification of nonreplicators and where they hide within the host have significant implications for the way novel chemotherapeutic agents and regimens are designed for persistent infections. [ABSTRACT FROM AUTHOR]

The ability to initiate DNA replication is a critical step in the proliferation of all organisms. In bacteria, this process is mediated by an ATP‐dependent replication initiator protein, DnaA, which recognizes and melts replication origin (oriC) elements. Despite decades of biochemical and structural work, a mechanistic understanding of how DnaA recognizes and unwinds oriC has remained enigmatic. A recent study by Pelliciari et al. provides important new structural insights into how DnaA from Bacillus subtilis recognizes and processes its cognate oriC, showing how DnaA uses sequence features encoded in the origin to engage melted DNA. Comparison of the DnaA‐oriC structure with archaeal/eukaryl replication origin complexes based on Orc‐family proteins reveals a high degree of similarity in origin engagement by initiators from di domains of life, despite fundamental differences in origin melting mechanisms. These findings provide valuable insights into bacterial replication initiation and highlight the intriguing evolutionary history of this fundamental biological process. [ABSTRACT FROM AUTHOR]

Finalization of eukaryotic nuclear DNA replication relies on DNA ligase 1 (LIG1) to seal DNA nicks generated during Okazaki Fragment Maturation (OFM). Using a mutational reporter in Saccharomyces cerevisiae, we previously showed that mutation of the high-fidelity magnesium binding site of LIG1Cdc9 strongly increases the rate of single-base insertions. Here we show that this rate is increased across the nuclear genome, that it is synergistically increased by concomitant loss of DNA mismatch repair (MMR), and that the additions occur in highly specific sequence contexts. These discoveries are all consistent with incorporation of an extra base into the nascent lagging DNA strand that can be corrected by MMR following mutagenic ligation by the Cdc9-EEAA variant. There is a strong preference for insertion of either dGTP or dTTP into 3–5 base pair mononucleotide sequences with stringent flanking nucleotide requirements. The results reveal unique LIG1Cdc9-dependent mutational motifs where high fidelity DNA ligation of a subset of OFs is critical for preventing mutagenesis across the genome. DNA ligase 1 finalizes nuclear DNA replication by accurately sealing nicks generated during Okazaki Fragment Maturation. Here, Williams et al employ a low fidelity DNA ligase 1 variant to study mutation rates and specificity across the yeast genome [ABSTRACT FROM AUTHOR]

Skin macrophages play important roles in the response to external stimuli. Human skin equivalents (HSEs) incorporating the human monocytic cell line THP-1 were fabricated to generate immunocompetent human skin models. These HSEs were used to investigate the influence of the skin microenvironment and ultraviolet A (UVA) on macrophages. Transcriptomic analysis revealed that THP-1 cells in HSEs were enriched in extracellular matrix interaction hallmark but downregulated in DNA replication hallmark. Upon UVA exposure, immunocompetent HSEs presented epidermal distortion and increased DNA double-strand breaks (DSBs). The genes associated with oxidative stress and the inflammatory response were significantly upregulated in THP-1 cells. When the photoprotective agent mycosporine-2-glycine from cyanobacteria was applied to HSEs, the incidence of UVA-induced DSBs was significantly lower, and inflammatory and UV response hallmarks were downregulated in THP-1 cells. Taken together, these results suggest that immunocompetent HSEs can be used to investigate the responses of skin-resident macrophages to external stimuli. Human skin equivalents respond to UV stimulation by upregulating inflammatory and oxidative stress pathways in macrophages, representing a model to study skin immune responses to external stimuli. [ABSTRACT FROM AUTHOR]

Force-driven cellular interactions are crucial for cancer cell invasion but remain underexplored in vascular abnormalities. Cerebral cavernous malformations (CCM), a vascular abnormality characterized by leaky vessels, involves CCM mutant cells recruiting wild-type endothelial cells to form and expand mosaic lesions. The mechanisms behind this recruitment remain poorly understood. Here, we use an in-vitro model of angiogenic invasion with traction force microscopy to reveal that hyper-angiogenic Ccm2-silenced endothelial cells enhance angiogenic invasion of neighboring wild-type cells through force and extracellular matrix-guided mechanisms. We demonstrate that mechanically hyperactive CCM2-silenced tips guide wild-type cells by transmitting pulling forces and by creating paths in the matrix, in a ROCKs-dependent manner. This is associated with reinforcement of β1 integrin and actin cytoskeleton in wild-type cells. Further, wild-type cells are reprogrammed into stalk cells and activate matrisome and DNA replication programs, thereby initiating proliferation. Our findings reveal how CCM2 mutants hijack wild-type cell functions to fuel lesion growth, providing insight into the etiology of vascular malformations. By integrating biophysical and molecular techniques, we offer tools for studying cell mechanics in tissue heterogeneity and disease progression. In cerebral cavernous malformations, mutant cells recruit healthy endothelial cells to form mosaic lesions. Here, the authors show that mutant cells can hijack and reprogram neighboring wildtype cells by mechanically pulling on them. [ABSTRACT FROM AUTHOR]

Mcm10 plays an essential role in the activation of replicative helicase CMG through the cell cycle-regulated interaction with the prototype MCM double hexamer in Saccharomyces cerevisiae. In this study, we reported that Mcm10 is phosphorylated by S-phase cyclin-dependent kinases (S-CDKs) at S66, which enhances Mcm10--MCM association during the S phase. S66A single mutation or even deletion of whole N-terminus (a.a. 1-128) only causes mild growth defects. Nevertheless, S66 becomes indispensable in the absence of the Mcm10 C-terminus ((a.a. 463-571), the major MCM-binding domain. Using a two-degron strategy to efficiently deplete Mcm10, we show that mcm10-S66AΔC has a severe defect in proceeding into the S phase. Notably, both lethality and S-phase deficiency can be rescued by artificially tethering mcm10-S66AΔC to MCM. These findings illustrate how the Mcm10-MCM association is regulated as a crucial event in DNA replication initiation. [ABSTRACT FROM AUTHOR]

The core structure is an aromatic heterocyclic organic compound i.e. Isoquinoline. Both the structures of Isoquinoline and quinoline contain a fused ring structure of benzene and pyridine. Isoquinoline, the pyridine counterpart, has a pKb of 8.6 and a pKa of 5.14, making it a weak basic. When it comes into contact with Lewis acids, such BF3, it protonates and becomes a salt. This process produces adducts. The liquid form of Isoquinoline is colorless, hygroscopic, and it has an unpleasant smell. Isoquinoline derivatives are a very significant group of natural and synthetic compounds which show various Pharmacological activities like anti-cancer, anti-oxidant, anti-microbial, anti-inflammatory, anti-microbial, analgesic, anti-fungal, anti-viral, antispasmodic and an enzyme inhibitor. Morphine and codeine are the most extensively characterized Isoquinoline alkaloids. They are made from either tyrosine or phenylalanine. Some of the Isoquinoline nucleus-containing drugs available in the market were Nelfinavir, Apomorphine, Quinapril, Praziquantel, Solifenacin, Papaverine, Metocurine, Tubocurarine. Isoquinoline alkaloids with anticancer properties may be therapeutically to target specific binding to nucleic acids, modulating polynucleic acid stability. These binds change the way that duplex B-form DNA interacts with proteins involved in DNA replication, repair, or transcription. [ABSTRACT FROM AUTHOR]

Objectives: Acute temperature stress was explored in Hippocampus abdominalis through a comprehensive RNA-seq analysis. Methods: RNA-seq was conducted on 20-day-old H. abdominalis after 24 h of temperature stress. Four experimental conditions were established: a control group (18 °C) and three temperature treatment groups (21, 24, and 27 °C). Results: Seahorse larvae were found to be unaffected by 21 °C and 24 °C and were able to survive for short periods of time during 24 h of incubation, whereas mortality approached 50% at 27 °C. The sequencing process produced 75.63 Gb of high-quality clean data, with Q20 and Q30 base percentages surpassing 98% and 96%, respectively. A total of 141, 333, and 1598 differentially expressed genes were identified in the 21, 24, and 27 °C groups vs. a control comparison group, respectively. Notably, the number of up-regulated genes was consistently higher than that of down-regulated genes across all comparisons. Gene Ontology functional annotation revealed that differentially expressed genes were predominantly associated with metabolic processes, redox reactions, and biosynthetic functions. In-depth KEGG pathway enrichment analysis demonstrated that down-regulated genes were significantly enriched in pathways related to steroid biosynthesis, terpenoid backbone biosynthesis, spliceosome function, and DNA replication. Up-regulated genes were enriched in pathways associated with the FoxO signaling pathway and mitophagy (animal). The results indicated that temperature stress induced extensive changes in gene expression in H. abdominalis, involving crucial biological processes such as growth, biosynthesis, and energy metabolism. Conclusions: This study provided key molecular mechanisms in the response of H. abdominalis to temperature stress, offering a strong basis for future research aimed at understanding and mitigating the effects of environmental stressors on marine species. [ABSTRACT FROM AUTHOR]

Epstein-Barr virus (EBV) co-infections with human papillomavirus (HPV) have been observed in oropharyngeal squamous cell carcinoma. Modeling EBV/HPV co-infection in organotypic epithelial raft cultures revealed that HPV16 E7 inhibited EBV productive replication through the facilitated degradation of the retinoblastoma protein pRb/p105. To further understand how pRb is required for EBV productive replication, we generated CRISPR-Cas9 pRb knockout (KO) normal oral keratinocytes (NOKs) in the context of wild-type and mutant K120E p53. EBV replication was examined in organotypic rafts as a physiological correlate for epithelial differentiation. In pRb KO rafts, EBV DNA copy number was statistically decreased compared to vector controls, regardless of p53 context. Loss of pRb did not affect EBV binding or internalization of calcium-treated NOKs or early infection of rafts. Rather, the block in EBV replication correlated with impaired immediate early gene expression. An EBV infection time course in rafts with mutant p53 demonstrated that pRb-positive basal cells were initially infected with delayed replication occurring in differentiated layers. Loss of pRb showed increased S-phase progression makers and elevated activator E2F activity in raft tissues. Complementation with a panel of pRb/E2F binding mutants showed that wild type or pRbÎ"685 mutant capable of E2F binding reduced S-phase marker gene expression, rescued EBV DNA replication, and restored BZLF1 expression in pRb KO rafts. However, pRb KO complemented with pRb661W mutant, unable to bind E2Fs, failed to rescue EBV replication in raft culture. These findings suggest that EBV productive replication in differentiated epithelium requires pRb inhibition of activator E2Fs to restrict S-phase progression. [ABSTRACT FROM AUTHOR]