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Protein–protein interactions (PPIs) in the nucleus play key roles in transcriptional regulation and ensure genomic stability. Critical to this are histone-mediated PPI networks, which are further fine-tuned through dynamic post-translational modification. Perturbation to these networks leads to genomic instability and disease, presenting epigenetic proteins as key therapeutic targets. This mini-review will describe progress in mapping the combinatorial histone PTM landscape, and recent chemical biology approaches to map histone interactors. Recent advances in mapping direct interactors of histone PTMs as well as local chromatin interactomes will be highlighted, with a focus on mass-spectrometry based workflows that continue to illuminate histone-mediated PPIs in unprecedented detail.

Neurodegeneration is a complex neurological phenomenon characterized by disturbed coherence in neuronal efflux. Progressive neuronal loss and brain damage due to various age-related pathological hallmarks perturb the behavioral balance and quality of life. Sirtuins have been widely investigated for their neuroprotective role, with SIRT1 being the most contemplated member of the family. SIRT1 exhibits significant capabilities to enhance neurogenesis and cellular lifespan by regulating various pathways, which makes it an exciting therapeutic target to inhibit neurodegenerative disease progression. SIRT1 mediated neuronal fortification involves modulation of molecular co-factors and biochemical pathways responsible for the induction and sustenance of pro-inflammatory and pro-oxidative environment in the cellular milieu. In this review, we present the major role played by SIRT1 in maintaining cellular strength through the regulation of genomic stability, neuronal growth, energy metabolism, oxidative stress, inhibiting mechanisms and anti-inflammatory responses. The therapeutic significance of SIRT1 has been put into perspective through a comprehensive discussion about its ameliorating potential against neurodegenerative stimuli in a variety of diseases that characteristically impair cognition, memory and motor coordination. This review enhances the acquaintance concerned with the neuroprotective potential of SIRT1 and thus promotes the development of novel SIRT1 regulating therapeutic agents and strategies.

BackgroundMillions of workers worldwide are exposed to diesel engine exhaust (DEE), a known genotoxic carcinogen. Alu retroelements are repetitive DNA sequences that can multiply and compromise genomic stability. There is some evidence linking altered Alu repeats to cancer and elevated mortality risks. However, whether Alu repeats are influenced by environmental pollutants is unexplored. In an occupational setting with high DEE exposure levels, we investigated associations with Alu repeat copy number.MethodsA cross-sectional study of 54 male DEE-exposed workers from an engine testing facility and a comparison group of 55 male unexposed controls was conducted in China. Personal air samples were assessed for elemental carbon, a DEE surrogate, using NIOSH Method 5040. Quantitative PCR (qPCR) was used to measure Alu repeat copy number relative to albumin (Alb) single-gene copy number in leucocyte DNA. The unitless Alu/Alb ratio reflects the average quantity of Alu repeats per cell. Linear regression models adjusted for age and smoking status were used to estimate relations between DEE-exposed workers versus unexposed controls, DEE tertiles (6.1–39.0, 39.1–54.5 and 54.6–107.7 µg/m3) and Alu/Alb ratio.ResultsDEE-exposed workers had a higher average Alu/Alb ratio than the unexposed controls (p=0.03). Further, we found a positive exposure–response relationship (p=0.02). The Alu/Alb ratio was highest among workers exposed to the top tertile of DEE versus the unexposed controls (1.12±0.08 SD vs 1.06±0.07 SD, p=0.01).ConclusionOur findings suggest that DEE exposure may contribute to genomic instability. Further investigations of environmental pollutants, Alu copy number and carcinogenesis are warranted.

The objective of the study: a comparative analysis of genome-wide sequences of seed lot (SL) 361 "sh" M. bovis BCG-1 (Russia) and its daughter isolates obtained from children with manifestations of BCG osteitis.Subjects and methods: genotyping; bioinformational analysis of genome-wide sequences of PS 361 "sh” vaccine BCG-1 (Russia) and its daughter isolates.Results. PS 361 "sh" BCG-1 (Russia) was heterogeneous in 7 coding sequences with different ratios of single nucleotide substitutions inherited by 2 of 6 daughter BCG isolates. At the same time, polymorphisms did not violate the genomic stability and viability of the BCG vaccine during long-term existence in vivo. In isolates of BCG 2925 and 5448, accumulation (hypothetically, under the pressure of selection in the vaccinated organism) of nonsynonymous substitutions in 4 out of 7 polymorphic genes was noted, including the genes ppsC, eccD5, and eccA5 associated with mycobacterial virulence. Evaluation of the significance of genomic variations in BCG isolates relating to the association with the development of post-vaccination osteitis requires more detailed information about the immune status of patients. Genome-wide analysis of production strains, seed lots, finished vaccine lots and daughter clinical isolates makes the contemporary approach to understanding the molecular basis of the efficacy and complications of BCG vaccination.

In this study, arsenic (As)-induced genotoxicity in the roots and shoots of Cucumis melo (melon) seedlings were investigated by using the random amplified polymorphic DNA - polymerase chain reaction (RAPD-PCR) and comet assays. For this purpose, melon seedlings were exposed to arsenate [As(V)] at 0, 100, 200, 300, and 400 μM concentrations in the hydroponic system for 14 days to examine the level of As accumulation, alterations in growth parameters, and DNA damage. A reduction in growth with increasing As(V) concentration was observed in the melon seedlings. Total As accumulations in the shoot and root tissue increased in a dose-dependent manner; however, the level was higher in the roots than the shoots. In RAPD-PCR analysis, 26 primers gave reproducible and scorable results and produced a total of 128 bands in the control seedlings. Alterations in RAPD profiles, including the loss or appearance of new bands, were determined in the As-treated seedlings when compared to the control. The values of genomic template stability (GTS) were decreased by increasing the concentration of the As(V) in both tissue types. DNA strand breaks were observed in all the tested As(V) concentrations in the alkaline comet assay; furthermore, the loss of DNA integrity was higher with 300 and 400 μM As(V) treatments. The results clearly indicate that the combination of DNA-based molecular and cytogenetic techniques (e.g. the comet assay) may be proposed as a reliable evaluation of genotoxicity in plants after exposure to heavy metal pollution.

Background Flap endonuclease 1 (FEN1) is a structure-specific nuclease that plays a role in a variety of DNA metabolism processes. FEN1 is important for maintaining genomic stability and regulating cell growth and development. It is associated with the occurrence and development of several diseases, especially cancers. There is a lack of systematic bibliometric analyses focusing on research trends and knowledge structures related to FEN1. Purpose To analyze hotspots, the current state and research frontiers performed for FEN1 over the past 15 years. Methods Publications were retrieved from the Web of Science Core Collection (WoSCC) database, analyzing publication dates ranging from 2005 to 2019. VOSviewer1.6.15 and Citespace5.7 R1 were used to perform a bibliometric analysis in terms of countries, institutions, authors, journals and research areas related to FEN1. A total of 421 publications were included in this analysis. Results Our findings indicated that FEN1 has received more attention and interest from researchers in the past 15 years. Institutes in the United States, specifically the Beckman Research Institute of City of Hope published the most research related to FEN1. Shen BH, Zheng L and Bambara Ra were the most active researchers investigating this endonuclease and most of this research was published in the Journal of Biological Chemistry. The main scientific areas of FEN1 were related to biochemistry, molecular biology, cell biology, genetics and oncology. Research hotspots included biological activities, DNA metabolism mechanisms, protein-protein interactions and gene mutations. Research frontiers included oxidative stress, phosphorylation and tumor progression and treatment. Conclusion This bibliometric study may aid researchers in the understanding of the knowledge base and research frontiers associated with FEN1. In addition, emerging hotspots for research can be used as the subjects of future studies.

The collapse of DNA replication forks leads to the formation of single-ended DNA double-strand breaks. One would have assumed that these breaks would be repaired as soon as possible. However, a recent study suggests that a delay in DNA polymerase θ-mediated end joining until mitosis, orchestrated by BRCA2 and RAD52, favors genomic stability.

Graphical abstract, Long non-coding RNAs (lncRNAs) can perform a variety of key cellular functions by interacting with proteins and other RNAs. Recent studies have shown that the functions of lncRNAS are largely mediated by their structures. However, our structural knowledge for most lncRNAS is limited to sequence-based computational predictions. Non-coding RNA activated by DNA damage (NORAD) is an atypical lncRNA due to its abundant expression and high sequence conservation. NORAD regulates genomic stability by interacting with proteins and microRNAs. Previous sequence-based characterization has identified a modular organization of NORAD composed of several NORAD repeat units (NRUs). These units comprise the protein-binding elements and are separated by regular spacers. Here, we experimentally determine for the first time the secondary structure of NORAD using the nextPARS approach. Our results suggest that the spacer regions provide structural stability to NRUs. Furthermore, we uncover two previously unreported NRUs, and determine the core structural motifs conserved across NRUs. Overall, these findings will help to elucidate the function and evolution of NORAD.

Sumoylation is an essential post-translational modification intimately involved in a diverse range of eukaryotic cellular mechanisms. Small ubiquitin-like modifier (SUMO) protein isoforms can be reversibly linked to lysine residues that reside within specific motifs on thousands of target substrates, leading to modulations in stability, solubility, localization, and interactor profile. Since its initial discovery almost 25 years ago, SUMO has been described as a key regulator of genomic stability, cell proliferation, and infection among other processes. In this review, we trace the exciting developments in the history of this critical modifier, highlighting SUMO's roles in pathogenesis as well as its potential for the development of targeted therapies for numerous diseases.

Telomere attrition and mitochondrial DNA variations are implicated in the biological aging process and genomic stability can be influenced by nutritional factors. This study aims to analyze the relative telomere length (rTL) and mitochondrial DNA copy number (mtCN) in aged individuals and their association with plasma folate and vitamin B12 levels. This community-based cross-sectional study involves 428 subjects (60 years: 242≥60 years: 186). Quantitative real-time PCR was used to measure rTL and mtCN variation, and radioimmunoassay to measure plasma folate and vitamin B12 levels. The subjects in the ≥60 years age group have significantly shorter telomeres and lower mtCN compared to the60 years age group. A significant positive correlation was observed between the rTL and mtCN, and both of them were positively associated with plasma folate and vitamin B12 levels. In the ≥60 age group; folate and vitamin B12 positively correlated with rTL and vitamin B12 with mtCN. The study revealed a decline of rTL and mtCN with age in the Indian population and their association suggests that they may co-regulate each other with age. In conclusion, folate and vitamin B12 may delay aging by preventing the reduction in rTL length and mtCN.

BRCA2 is a key breast cancer associated protein that is predicted to have interspersed regions of intrinsic disorder. Intrinsic disorder coupled with large size likely allows BRCA2 to sample a broad range of conformational space. We expect that the resulting dynamic arrangements of BRCA2 domains are a functionally important aspect of its role in homologous recombination DNA repair. To determine the architectural organization and the associated conformational landscape of BRCA2, we used scanning force microscopy based single molecule analyses to map the flexible regions of the protein and characterize which regions influence oligomerization. We show that the N- and the C-terminal regions are the main flexible regions. Both of these regions also influence BRCA2 oligomerization and interaction with RAD51. In the central Brc repeat region, Brc 1–4 and Brc 5–8 contribute synergistically to BRCA2 interaction with RAD51. We also analysed several single amino acid changes that are potentially clinically relevant and found one, the variant of F1524V, which disrupts key interactions and alters the conformational landscape of the protein. We describe the overall conformation spectrum of BRCA2, which suggests that dynamic structural transitions are key features of its biological function, maintaining genomic stability.

Pluripotent stem cells (PSCs) are capable of generating all types of cells in the body and have promising applications in basic research and cell-based regenerative medicine. Compared to the differentiated cells, PSCs are superior in maintaining genomic stability. However, the underlying molecular mechanisms are far from clear. Here, we summarized the understandings on the molecules and pathways that PSCs specifically utilize to cope with DNA replication-associated stress, to repair DNA damages and to determine cell fates.

Telomeres are genetically conserved nucleoprotein complexes located at the ends of chromosomes that preserve genomic stability. In large mammals, somatic cell telomeres shorten with age, owing to the end replication problem and lack of telomere-lengthening events (e.g. telomerase and ALT activity). Therefore, telomere length reflects cellular replicative reserve and mitotic potential. Environmental insults can accelerate telomere attrition in response to cell division and DNA damage. As such, telomere shortening is considered one of the major hallmarks of ageing. Much effort has been dedicated to understanding the environmental perturbations that accelerate telomere attrition and therapeutic strategies to preserve or extend telomeres. As telomere dynamics seem to reflect cumulative cellular stress, telomere length could serve as a biomarker of animal welfare. The assessment of telomere dynamics (i.e. rate of shortening) in conjunction with telomere-regulating genes and telomerase activity in racehorses could monitor long-term animal health, yet it could also provide some unique opportunities to address particular limitations with the use of other animal models in telomere research. Considering the ongoing efforts to optimise the health and welfare of equine athletes, the purpose of this review is to discuss the potential utility of assessing telomere length in Thoroughbred racehorses. A brief review of telomere biology in large and small mammals will be provided, followed by discussion on the biological implications of telomere length and environmental (e.g. lifestyle) factors that accelerate or attenuate telomere attrition. Finally, the utility of quantifying telomere dynamics in horses will be offered with directions for future research.

As the primary catalyst of homologous recombination (HR) in vertebrates, RAD51 has been extensively studied in the context of repair of double-stranded DNA breaks (DSBs). With recent advances in the understanding of RAD51 function extending beyond DSBs, the importance of RAD51 throughout DNA metabolism has become increasingly clear. Here we review the suggested roles of RAD51 beyond HR, specifically focusing on their interplay with DNA replication and the maintenance of genomic stability, in which RAD51 function emerges as a double-edged sword.

Glioblastoma (GBM) is the most aggressive and common type of adult malignant brain tumor, with a median survival of only 21 months. To identify which genes drive its highly aggressive phenotype, we performed a genome-wide CRISPR-Cas9 knockout screen. Results showed substantial enrichment of ~160 novel essential oncogenic driver genes and pathways, including a previously unstudied gene THOC1—involved in RNA processing—that showed significant elevations in expression at RNA and protein levels (p< 0.05) in GBM, as well as a significant survival benefit in patient datasets when downregulated (p< 0.05). Knocking out THOC1 resulted in cell death in multiple GBM patient-derived xenograft (PDX) lines and extended survival compared to the controls (p< 0.01) in vivo. Overexpression of THOC1 in neural stem cells resulted in transformation to a cancerous phenotype, as evidenced by sphere formation in a soft agar assay (p< 0.01) and in vivo tumor engraftment assays. Further investigation of THOC1 through immunoprecipitation in neural stem cells and multiple GBM lines showed significant interaction in GBM with histone deacetylase complex SIN3A, involved in recruiting major histone deacetylases in order to close the DNA and prevent the accumulation of R-loops, RNA:DNA hybrids that pose a threat to genomic stability. Additional investigation revealed that THOC1-knockdowns in vitro induced R-loop formation and DNA damage, while THOC1-overexpression in vitro resulted in an untenable decrease in R-loops and DNA damage, suggesting that the THOC1-SIN3A axis is elevated in GBM in order to prevent the accumulation of genotoxic R-loops. Additionally, histone deacetylase activity was shown to be elevated in THOC1-overexpression conditions and reduced in THOC1-knockdown conditions, confirming that the THOC1-SIN3A axis functions to prevent R-loop accumulation through the epigenetic regulation. In summary, our whole-genome CRISPR-Cas9 knockout screen has identified a promising therapeutic target for GBM—a disease desperately in need of therapeutic innovations.

Remarkably, the p53 transcription factor, referred to as “the guardian of the genome”, is not essential for mammalian development. Moreover, efforts to identify p53-dependent developmental events have produced contradictory conclusions. Given the importance of pluripotent stem cells as models of mammalian development, and their applications in regenerative medicine and disease, resolving these conflicts is essential. Here we attempt to reconcile disparate data into justifiable conclusions predicated on reports that p53-dependent transcription is first detected in late mouse blastocysts, that p53 activity first becomes potentially lethal during gastrulation, and that apoptosis does not depend on p53. Furthermore, p53 does not regulate expression of genes required for pluripotency in embryonic stem cells (ESCs); it contributes to ESC genomic stability and differentiation. Depending on conditions, p53 accelerates initiation of apoptosis in ESCs in response to DNA damage, but cell cycle arrest as well as the rate and extent of apoptosis in ESCs are p53-independent. In embryonic fibroblasts, p53 induces cell cycle arrest to allow repair of DNA damage, and cell senescence to prevent proliferation of cells with extensive damage.

Glioblastoma (GBM) is the most common aggressive primary malignant brain tumor, and patients with GBM have a median survival of 20 months. Clinical therapy resistance is a challenging barrier to overcome. Tumor genome stability maintenance during DNA replication, especially the ability to respond to replication stress, is highly correlated with drug resistance. Recently, we identified a protective role for RECQ1 under replication stress conditions. RECQ1 acts at replication forks, binds PCNA, inhibits single-strand DNA formation and nascent strand degradation in GBM cells. It is associated with the function of the PARP1 protein, promoting PARP1 recruitment to replication sites. RECQ1 is essential for DNA replication fork protection and tumor cell proliferation under replication stress conditions, and as a target of RECQ1, PARP1 effectively protects and restarts stalled replication forks, providing new insights into genomic stability maintenance and replication stress resistance. These findings indicate that tumor genome stability targeting RECQ1-PARP1 signaling may be a promising therapeutic intervention to overcome therapy resistance in GBM.

Breast Cancer gene 1 (BRCA1) is a large, multi-functional protein that regulates a variety of mechanisms in multiple different tissues. Our work established that Brca1 is expressed in skeletal muscle and localizes to the mitochondria and nucleus. Here, we propose BRCA1 expression is critical for the maintenance of force production and mitochondrial respiration in skeletal muscle.

Protection of genome integrity is vital for all living organisms, particularly when DNA double-strand breaks (DSBs) occur. Eukaryotes have developed two main pathways, namely Non-Homologous End Joining (NHEJ) and Homologous Recombination (HR), to repair DSBs. While most of the current research is focused on the role of key protein players in the functional regulation of DSB repair pathways, accumulating evidence has uncovered a novel class of regulating factors termed non-coding RNAs. Non-coding RNAs have been found to hold a pivotal role in the activation of DSB repair mechanisms, thereby safeguarding genomic stability. In particular, long non-coding RNAs (lncRNAs) have begun to emerge as new players with vast therapeutic potential. This review summarizes important advances in the field of lncRNAs, including characterization of recently identified lncRNAs, and their implication in DSB repair pathways in the context of tumorigenesis.

The Knl1-Mis12-Ndc80 (KMN) network is an essential component of the kinetochore–microtubule attachment interface, which is required for genomic stability in eukaryotes. However, little is known about plant Knl1 proteins because of their complex evolutionary history. Here, we cloned the Knl1 homolog from maize (Zea mays) and confirmed it as a constitutive central kinetochore component. Functional assays demonstrated their conserved role in chromosomal congression and segregation during nuclear division, thus causing defective cell division during kernel development when Knl1 transcript was depleted. A 145 aa region in the middle of maize Knl1, that did not involve the MELT repeats, was associated with the interaction of spindle assembly checkpoint (SAC) components Bub1/Mad3 family proteins 1 and 2 (Bmf1/2) but not with the Bmf3 protein. They may form a helical conformation with a hydrophobic interface with the TPR domain of Bmf1/2, which is similar to that of vertebrates. However, this region detected in monocots shows extensive divergence in eudicots, suggesting that distinct modes of the SAC to kinetochore connection are present within plant lineages. These findings elucidate the conserved role of the KMN network in cell division and a striking dynamic of evolutionary patterns in the SAC signaling and kinetochore network.

Triple‐negative breast cancer (TNBC) is a malignancy that currently lacks targeted therapies. The majority of TNBCs can be characterized as basal‐like and has an expression profile enriched with genes involved in DNA damage repair and checkpoint response. Here, we report that TNBC cells are under replication stress and are constantly generating DNA double‐strand breaks, which is not seen in non‐TNBC cells. Consequently, we found that RECQL5, which encodes a RecQ family DNA helicase involved in many aspects of DNA metabolism including replication and repair, was essential for TNBC cells to survive and proliferate in vitro and in vivo. Compromising RECQL5 function in TNBC cells results in persistence of DNA damage, G2 arrest, and ultimately, cessation of proliferation. Our results suggest RECQL5 may be a potential therapeutic target for TNBC.

Poly(ADP-ribosyl)ation (PARylation) is a reversible post-translational modification regulating various biological pathways including DNA damage repair (DDR). Rapid turnover of PARylation is critically important for an optimal DNA damage response and maintaining genomic stability. Recent studies show that PARylation is tightly regulated by a group of enzymes that can erase the ADP-ribose (ADPR) groups from target proteins. The aim of this review is to present a comprehensive understanding of dePARylation enzymes, their substrates and roles in DDR. Special attention will be laid on the role of these proteins in the development of cancer and their feasibility in anticancer therapeutics.

Polyploidization, also whole-genome duplication (WGD), is ubiquitous in plants but in vertebrates it occurs relatively rarely. The mechanisms that allow polyploid plants to overcome “genome shock” and achieve genomic stability, and how doubled genomes evolve had been well characterized. These studies yield several models that serve as theoretical references for analyses of polyploid vertebrates. Herein, we review the development of theories in WGD studies, summarize the research models and progress in vertebrates, and discuss the challenges of “omics” research and effective technical strategies. We hope to discover feasible means of unravelling the genome evolution of polyploid vertebrates.

One of the most important environmental pollutants is cement dust. In this study, RAPD technology was used todefine the potential genotoxic impact of cement dust on plants. For this purpose, the DNA comparison of 12 plant species(Convolvulus sepium, Astragalus christianus, Taraxacum androssovii Medicago varia, Alyssum murale, Artemisiaspisigera, Falcaria vulgaris, Anchusa strigose, Glaucium leiocarpum, Salvia syriaca, Cryciata taurica Tragopogonalbinervis)collected from the areas of 10000 m (Control area) or 0-100 m away from Askale cement factory in Erzurum;was performed. 16 primers were used, and 390 bands were obtained. Important differences were observed in plant RAPDprofiles collected from 0-100 m areas when compared to their respective controls (10000 m away from the factory). Somebands in control plants got lost, and new band formations were observed. The Genomic template stability (GTS) valuedecreased significantly in plants collected from 0-100 m area as compared to the controls (%56.20). Soil samples from 0-100 m and control area were collected and examined for their heavy metal contents. According to the analysis results,Nickel, Cadmium, Zinc, Lead and Copper concentrations were found to be dramatically high in the soil collected from 0-100 m as compared to the control area. These results indicate that the high heavy metal content in the soil causes thechanges in RAPD profiles and GTS in plants. The data also indicated that RAPD and GTS assays are basic, effective andreproducible means of the genotoxicity control of environmental pollution. En önemli çevre kirleticilerinden biri çimento tozudur. Bu çalışmada, çimento tozunun bitkiler üzerindekipotansiyel genotoksik etkisini tanımlamak için RAPD teknolojisi kullanılmıştır. Bu amaçla, Erzurum'da Askale çimentofabrikasına 10000 m (Kontrol alanı) ve 0-100 m uzaklıkta toplanan 12 bitki türünün ( Convolvulus sepium, Astragaluschristianus, Taraxacum androssovii Medicago varia, Alyssum murale, Artemisia spisigera, Falcaria vulgaris, Anchusastrigose, Glaucium leiocarpum, Salvia syriaca, Cryciata taurica Tragopogon albinervis)DNA karşılaştırması yapıldı.Çalışmada 16 RAPD primeri kullanılmış ve 390 bant elde edilmiştir. 0-100m alanlardan toplanan bitki RAPDprofillerinde, kontrollerine göre (fabrikadan 10000 metre uzakta) önemli farklılıklar gözlenmiştir. Kontrol bitkilerindebazı bantların kaybolduğu ve yeni bantların oluştuğu görülmüştür. Ayrıca, GTS (Genomic template stability) değeri, 0-100 m alandan toplanan bitkilerde, kontrollerinkilerle karşılaştırıldığında önemli ölçüde azaldığı görülmüştür( %56.20).0-100m ve kontrol alanından toprak örnekleri toplanmış ve ağır metal içerikleri bakımından incelenmiştir. Analizsonuçlarına göre, 0-100 m'den toplanan topraklarda Nikel, Kadmiyum, Çinko, Kurşun ve Bakır konsantrasyonlarının,çarpıcı şekilde yüksek olduğu bulunmuştur. Bu sonuçlar topraktaki yüksek ağır metal içeriğinin bitkilerde RAPDprofillerinde ve GTS’de değişikliklere neden olduğunu göstermektedir. Sonuçlar RAPD ve GTS testlerinin çevre kirliliğinin bitkiler üzerindeki genotoksik etkisini tanımlamak için temel, etkili ve tekrarlanabilir yöntemler olduğunugöstermiştir.

The aim of the present work was to determine proliferation capacity, immunophenotype and genome integrity of mesenchymal stromal cells (MSCs) from horse umbilical cord blood (UCB) at passage stage 5 and 10. Passage 4 cryopreserved UCB-MSCs from six unrelated donors were evaluated. Immunophenotypic analysis of UCB-MSC revealed a cell identity consistent with equine MSC phenotype by high expression of CD90, CD44, CD29, and very low expression of CD4, CD11a/18, CD73, and MHC class I and II antigens. Proliferative differences were noted among the UCB-MSC cultures. UCB-MSCs karyotype characteristics at passage 5 (eg, 2n = 64; XY, or XX) included 20% polyploidy and 62% aneuploidy. At passage 10, the proportion of polyploidy and aneuploidy was 21% and 82%, respectively, with the increase in aneuploidy being significant compared with passage 5. Furthermore, conventional GTG-banded karyotyping revealed several structural chromosome abnormalities at both passage 5 and 10. The clinical relevance of such chromosome instability is unknown, but determination of MSC cytogenetic status and monitoring of patient response to MSC therapies would help address this question.

Apart from its well-documented role in long-term promoter silencing, the genome-wide distribution patterns of ~ 28 million methylated or unmethylated CpG dinucleotides, e. g. in the human genome, is in search of genetic functions. We have set out to study changes in the cellular CpG methylation profile upon introducing foreign DNA into mammalian cells. As stress factors served the genomic integration of foreign (viral or bacterial plasmid) DNA, virus infections or the immortalization of cells with Epstein Barr Virus (EBV). In all instances investigated, alterations in cellular CpG methylation and transcription profiles were observed to different degrees. In the case of adenovirus DNA integration in adenovirus type 12 (Ad12)-transformed hamster cells, the extensive changes in cellular CpG methylation persisted even after the complete loss of all transgenomic Ad12 DNA. Hence, stress-induced alterations in CpG methylation can be inherited independent of the continued presence of the transgenome. Upon virus infections, changes in cellular CpG methylation appear early after infection. In EBV immortalized as compared to control cells, CpG hypermethylation in the far-upstream region of the human FMR1 promoter decreased four-fold. We conclude that in the wake of cellular stress due to foreign DNA entry, preexisting CpG methylation patterns were altered, possibly at specific CpG dinucleotides. Frequently, transcription patterns were also affected. As a working concept, we view CpG methylation profiles in mammalian genomes as a guarding sensor for genomic stability under epigenetic control. As a caveat towards manipulations of cells with foreign DNA, such cells can no longer be considered identical to their un-manipulated counterparts.

Most cells in the human body, including human mesenchymal stem cells (hMSCs), have evolved to survive and function in a low physiological oxygen (O2) environment. Investigators have become increasingly aware of the effects of O2 levels on hMSC biology and culture and are mimicking the natural niche of these cells in vitro to improve cell culture yields. This presents many challenges in relation to hMSC identity and function and in the maintenance of a controlled O2 environment for cell culture. The aim of this review was to discuss an "hMSC checklist" as a guide to establishing which identity and potency assays to implement when studying hMSCs. The checklist includes markers, differentiation potential, proliferation and growth, attachment and migration, genomic stability, and paracrine activity. Evidence drawn from the current literature demonstrates that low O2 environments could improve most "hMSC checklist" attributes. However, there are substantial inconsistencies around both the terminology and the equipment used in low O2 studies. Therefore, "hypoxia" as a term and as a culture condition is discussed. The biology of short-term (acute) versus long-term (chronic) hypoxia is considered, and a nascent hypothesis to explain the behavior of hMSCs in long-term hypoxia is presented. It is hoped that by establishing an ongoing discourse and driving toward a regulatory recognizable "hMSC checklist," we may be better able to provide the patient population with safe and efficacious regenerative treatments.

Recent studies have revealed the prevalence and biological significance of guanidine metabolism in nature. However, the metabolic pathways used by microbes to degrade guanidine or mitigate its toxicity have not been widely studied. Here we report a novel guanidine-degrading enzyme, Sll1077, identified in the model cyanobacterium Synechocystis sp. PCC 6803 through comparative proteomics and subsequent experimental validation. Although previously annotated as an agmatinase enzyme, Sll1077 is more likely a “guanidinase”, because it degrades guanidine rather than agmatine to urea. We demonstrate that the model cyanobacterium Synechococcus elongatus PCC 7942 strain engineered to express the bacterial ethylene-forming enzyme (EFE) exhibits unstable ethylene production due to toxicity and genomic instability induced by accumulation of the EFE-byproduct guanidine. Co-expression of EFE and Sll1077 significantly enhanced genomic stability and enabled the resulting strain to achieve sustained high-level ethylene production. These findings expand our knowledge of natural guanidine degradation pathways and demonstrate their biotechnological application to support ethylene bioproduction.

DNA helicases have emerged as a prominent class of nucleic acid-metabolizing enzymes that play important roles in genome maintenance and cellular homeostasis [...]

Proper functioning of cells—their ability to divide, differentiate, and regenerate—is dictated by genomic stability. The main factors contributing to this stability are the telomeric ends that cap chromosomes. Telomere biology and telomerase activity have been of interest to scientists in various medical science fields for years, including the study of both cancer and of senescence and aging. All these processes are accompanied by telomere-length modulation. Maintaining the key levels of telomerase component (hTERT) expression and telomerase activity that provide optimal telomere length as well as some nontelomeric functions represents a promising step in advanced anti-aging strategies, especially in dermocosmetics. Some known naturally derived compounds contribute significantly to telomere and telomerase metabolism. However, before they can be safely used, it is necessary to assess their mechanisms of action and potential side effects. This paper focuses on the metabolic potential of natural compounds to modulate telomerase and telomere biology and thus prevent senescence and skin aging.

Oxidative and alkylating DNA damage occurs under normal physiological conditions and exogenous exposure to DNA damaging agents. To counteract DNA base damage, cells have evolved several defense mechanisms that act at different levels to prevent or repair DNA base damage. Cells combat genomic lesions like these including base modifications, abasic sites, as well as single-strand breaks, via the base excision repair (BER) pathway. In general, the core BER process involves well-coordinated five-step reactions to correct DNA base damage. In this review, we will uncover the current understanding of BER mechanisms to maintain genomic stability and the biological consequences of its failure due to repair gene mutations. The malfunction of BER can often lead to BER intermediate accumulation, which is genotoxic and can lead to different types of human disease. Finally, we will address the use of BER intermediates for targeted cancer therapy.

Endocrine-driven malignancies, including breast and prostate cancer, are among the most common human cancers. The relationship between sex steroid hormones (eg, androgen, estrogen, and progesterone), their cognate receptors, and genomic stability lie at the center of endocrine-driven cancer development, progression, and therapeutic resistance. A variety of direct and indirect mechanisms have been described that link steroid hormone signaling to the loss of genomic integrity that drives early carcinogenesis. These effects are often enriched within endocrine receptor cistromes, accounting for the high proportion of mutations and rearrangements in the region of hormone response elements. In other cases, the effects are generalized and rely on a complex array of genetic, epigenetic, and metabolic interactions. Both androgen and estrogen receptors directly modulate the DNA damage response by trans-activating DNA damage response genes and redirecting the cellular repair machinery in the wake of genotoxic stress. Here we review the key mechanistic underpinnings of the relationship between sex steroid hormone receptors and genomic stability. In addition, we summarize emerging research in this area and discuss important implications for cancer prevention and treatment.

Eukaryotic genomes are compacted into loops and topologically associating domains (TADs), which contribute to transcription, recombination and genomic stability. Cohesin extrudes DNA into loops that are thought to lengthen until CTCF boundaries are encountered. Little is known about whether loop extrusion is impeded by DNA-bound macromolecular machines. We demonstrate that the replicative helicase MCM is a barrier that restricts loop extrusion in G1 phase. Single-nucleus Hi-C of one-cell embryos revealed that MCM loading reduces CTCF-anchored loops and decreases TAD boundary insulation, suggesting loop extrusion is impeded before reaching CTCF. Single-molecule imaging shows that MCMs are physical barriers that frequently constrain cohesin translocationin vitro.Simulations are consistent with MCMs as abundant, random barriers. We conclude that distinct loop extrusion barriers contribute to shaping 3D genomes.One Sentence SummaryMCM complexes are obstacles that impede the formation of CTCF-anchored loops.

The mouse mahoganoid mutation abrogating Mahogunin Ring Finger-1 (MGRN1) E3 ubiquitin ligase expression causes hyperpigmentation, congenital heart defects and neurodegeneration. To study the pathophysiology of MGRN1 loss, we compared Mgrn1-knockout melanocytes with genetically matched controls and melan-md1 (mahoganoid) melanocytes. MGRN1 knockout induced a more differentiated and adherent phenotype, decreased motility, increased the percentage of cells in the S phase of the cell cycle and promoted genomic instability, as shown by stronger &gamma, H2AX labelling, increased burden of DNA breaks and higher abundance of aneuploid cells. Lack of MGRN1 expression decreased the ability of melanocytes to cope with DNA breaks generated by oxidizing agents or hydroxyurea-induced replicative stress, suggesting a contribution of genomic instability to the mahoganoid phenotype. MGRN1 knockout in B16-F10 melanoma cells also augmented pigmentation, increased cell adhesion to collagen, impaired 2D and 3D motility and caused genomic instability. Tumors formed by Mgrn1-KO B16-F10 cells had lower mitotic indices, fewer Ki67-positive cells and showed a trend towards smaller size. In short-term lung colonization assays Mgrn1-KO cells showed impaired colonization potential. Moreover, lower expression of MGRN1 is significantly associated with better survival of human melanoma patients. Therefore, MGRN1 might be an important phenotypic determinant of melanoma cells.

TP53 binding protein 1 (53BP1) plays an important role in DNA damage repair and maintaining genomic stability. However, the mutations of 53BP1 in human cancers have not been systematically examined. Here, we have analyzed 541 somatic mutations of 53BP1 across 34 types of human cancer from databases of The Cancer Genome Atlas, International Cancer Genome Consortium and Catalogue of Somatic Mutations in Cancer. Among these cancer-associated 53BP1 mutations, truncation mutations disrupt the nuclear localization of 53BP1 thus abolish its biological functions in DNA damage repair. Moreover, with biochemical analyses and structural modeling, we have examined the detailed molecular mechanism by which missense mutations in the key domains causes the DNA damage repair defects. Taken together, our results reveal the functional defects of a set of cancer-associated 53BP1 mutations.

Global DNA hypomethylation is a characteristic feature of colorectal carcinoma (CRC). The tumor inhibitory effect of S-adenosylmethionine (SAM) methyl donor has been described in certain cancers including CRC. However, the molecular impact of SAM treatment on CRC cell lines with distinct genetic features has not been evaluated comprehensively. HT-29 and SW480 cells were treated with 0.5 and 1 mmol/L SAM for 48 h followed by cell proliferation measurements, whole-genome transcriptome and methylome analyses, DNA stability assessments and exome sequencing. SAM reduced cell number and increased senescence by causing S phase arrest, besides, multiple EMT-related genes (e.g., TGFB1) were downregulated in both cell lines. Alteration in the global DNA methylation level was not observed, but certain methylation changes in gene promoters were detected. SAM-induced &gamma, H2AX elevation could be associated with activated DNA repair pathway showing upregulated gene expression (e.g., HUS1). Remarkable genomic stability elevation, namely, decreased micronucleus number and comet tail length was observed only in SW480 after treatment. SAM has the potential to induce senescence, DNA repair, genome stability and to reduce CRC progression. However, the different therapeutic responses of HT-29 and SW480 to SAM emphasize the importance of the molecular characterization of CRC cases prior to methyl donor supplementation.

Ataxia with oculomotor apraxia type 2 (AOA2) is a slowly progressive, autosomal recessive disease characterized by the triad of ataxia, oculomotor apraxia, and sensorimotor neuropathy. The genetic basis of AOA2 is biallelic mutation of the SETX gene, resulting in reduced or absent senataxin, a DNA/RNA repair protein essential for genomic stability.In this case report, we described a case of AOA2 with two clear pathogenic SETX mutations, one of which is novel. We then discussed two further likely “in cis” SETX sequence changes (previously reported in the literature as pathogenic), and presented the case that they are likely benign polymorphisms.

Rho is a general transcription termination factor playing essential roles in RNA polymerase (RNAP) recycling, gene regulation, and genomic stability in most bacteria. Textbook models of transcription termination postulate that hexameric Rho loads onto RNA prior to contacting RNAP and then translocates along the transcript in pursuit of the moving RNAP to pull RNA from it. Here, we report the cryo-EM structures of two termination process intermediates. Prior to interacting with RNA, Rho forms a specific “pre-termination complex” (PTC) with RNAP and elongation factors NusA and NusG, which stabilize the PTC. RNA exiting RNAP interacts with NusA before entering the central channel of Rho from the distal C-terminal side of the ring. We map the principal interactions in the PTC and demonstrate their critical role in termination. Our results overturn the traditional termination models and support a mechanism in which the formation of a persistent PTC is a prerequisite for termination.

Telomeres are the ends of linear chromosomes comprised of repetitive nucleotide sequences in humans. Telomeres preserve chromosomal stability and genomic integrity. Telomere length shortens with every cell division in somatic cells, eventually resulting in replicative senescence once telomere length becomes critically short. Telomere shortening can be overcome by telomerase enzyme activity that is undetectable in somatic cells, while being active in germline cells, stem cells, and immune cells. Telomeres are bound by a shelterin complex that regulates telomere lengthening as well as protects them from being identified as DNA damage sites. Telomeres are transcribed by RNA polymerase II, and generate a long noncoding RNA called telomeric repeat-containing RNA (TERRA), which plays a key role in regulating subtelomeric gene expression. Replicative immortality and genome instability are hallmarks of cancer and to attain them cancer cells exploit telomere maintenance and telomere protection mechanisms. Thus, understanding the role of telomeres and their associated proteins in cancer initiation, progression and treatment is very important. The present review highlights the critical role of various telomeric components with recently established functions in cancer. Further, current strategies to target various telomeric components including human telomerase reverse transcriptase (hTERT) as a therapeutic approach in human malignancies are discussed.

Genomic perturbations due to inaccurate DNA replication, including inappropriate chromosomal segregation often underlie the development of cancer and neurodegenerative diseases. The incidence of these two diseases increases with age and exhibits an inverse association. Therefore, elderly subjects with cancer exhibit a reduced risk of a neurodegenerative disease, and vice versa. Both of these diseases are associated with aging and share several risk factors. Cells have multiple mechanisms to repair DNA damage and inaccurate replication. Previous studies have demonstrated that tumor suppressor proteins serve a critical role in the DNA damage response, which may result in genomic instability and thus induction of cellular apoptosis. Tumor suppressor genes, such as phosphatase and tensin homologue deleted on chromosome 10 (PTEN), breast cancer susceptibility gene 1 (BRCA1) and TP53 reduce genomic susceptibility to cancer by repairing the damaged DNA. In addition, these genes work cooperatively to ensure the inhibition of the development of several types of cancer. PTEN, BRCA1 and TP53 have been recognized as the most frequently deleted and/or mutated genes in various types of human cancer. Recently, tumor suppressor genes have also been shown to be involved in the development of neurodegenerative diseases. The present review summarizes the recent findings of the functions of these tumor suppressors that are associated with genomic stability, and are involved in carcinogenic and neurodegenerative cell signaling. A summary is presented regarding the interactions of these tumor suppressors with their partners which results in transduction of downstream signals. The implications of these functions for cancer and neurodegenerative disease-associated biology are also highlighted.

The target of rapamycin (TOR) protein kinase is at the core of growth factor- and nutrient-dependent signaling pathways that are well-known for their regulation of metabolism, growth, and proliferation. However, TOR is also involved in the regulation of gene expression, genomic and epigenomic stability. TOR affects nuclear functions indirectly through its activity in the cytoplasm, but also directly through active nuclear TOR pools. The mechanisms by which TOR regulates its nuclear functions are less well-understood compared with its cytoplasmic activities. TOR is an important pharmacological target for several diseases, including cancer, metabolic and neurological disorders. Thus, studies of the nuclear functions of TOR are important for our understanding of basic biological processes, as well as for clinical implications.

Objective The alphaproteobacterium Zymomonas mobilis is an efficient ethanol producer, and Z. mobilis-based biorefinery shows great potential for biofuel production. Serial co-cultivation is an emerging approach that promotes inter-species interactions which can improve or rewire the metabolic features in industrially useful microorganisms by inducing frequent mutations. We applied this method to assess if it improves or rewires the desirable physiological features of Z. mobilis, especially ethanol production. Results We performed serial co-culture of Z. mobilis with the baker’s yeast, Saccharomyces cerevisiae. We observed filamentation of Z. mobilis cells in the co-culture, indicating that the Z. mobilis cells were exposed to stress due to the presence of a competitor. After 50 times of serial transfers, we characterized the generated Z. mobilis strains, showing that long term co-culture did not drive significant changes in either the growth or profile of excreted metabolites in the generated strains. In line with this, whole genome sequencing of the generated Z. mobilis strains revealed only minor genetic variations from the parental strain. 50 generations of Z. mobilis monoculture did not induce morphological changes or any significant genetic variations. The result indicates that the method needs to be carefully optimized for Z. mobilis strain improvement. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

RECQ1 (also known as RECQL or RECQL1) belongs to the RecQ family of DNA helicases, members of which are linked with rare genetic diseases of cancer predisposition in humans. RECQ1 is implicated in several cellular processes, including DNA repair, cell cycle and growth, telomere maintenance, and transcription. Earlier studies have demonstrated a unique requirement of RECQ1 in ensuring chromosomal stability and suggested its potential involvement in tumorigenesis. Recent reports have suggested that RECQ1 is a potential breast cancer susceptibility gene, and missense mutations in this gene contribute to familial breast cancer development. Here, we provide a framework for understanding how the genetic or functional loss of RECQ1 might contribute to genomic instability and cancer.

The tremendous variation that exists between bacterial species illustrates the power of evolution, which is the continuous process of mutation and selection over time. Even within a bacterial species, individual members can harbour an impressive degree of genetic variation, depending on the species. The question then arises how similar the offspring of a given bacterial cell over time is, and how long it takes before differences are noticeable? Here we show that on the one hand one can expect random mutations to arise, as a result of various mechanisms. On the other hand, there are forces at play that keep the offspring of a cell genetically relatively constant, unless there is selection for a particular characteristic. The most common mechanisms behind mutations that can appear in a bacterial population are briefly introduced. Next, it is explained why nevertheless such mutations are rarely observed, as long as single colonies are randomly selected, unless selective pressures apply. Since quality control of industrial bacterial cultures is likely to depend heavily on genome sequencing in the near future, the accuracy of whole‐genomic sequencing technologies is also discussed. It can be concluded that the bacteriologists who started picking single colonies from agar plates more than hundred years ago were unknowingly ingeneous, as their practice maintains a bacterial culture stable over time. Significance and Impact of Study The questions addressed here are relevant for industries that depend on live bacteria for (manufacturing of) their products, as they have to guard their bacterial cultures that remain unchanged over time. The explanation why randomly selection of single colonies keeps a population stable can be of use in bacteriology courses. The limitations of whole‐genome sequencing are relevant to legislators to avoid overinterpretation of those data., Significance and Impact of Study: The questions addressed here are relevant for industries that depend on live bacteria for (manufacturing of) their products, as they have to guard their bacterial cultures that remain unchanged over time. The explanation why randomly selection of single colonies keeps a population stable can be of use in bacteriology courses. The limitations of whole‐genome sequencing are relevant to legislators to avoid overinterpretation of those data.

Fondazione Italiana per la Ricerca sul Cancro FIRC 18112 Sina Atashpaz.Fondazione Umberto Veronesi Sina Atashpaz Associazione Italiana per la Ricerca sul Cancro AIRC 5xmille METAMECH program Vincenzo Costanzo Giovanni Armenise-Harvard Foundation Vincenzo Costanzo European Research Council Consolidator grant 614541 Vincenzo Costanzo Associazione Italiana per la Ricerca sul Cancro Fellowship 23961 Negar ArghavanifarDanish Cancer Society KBVU-2014 Andres Joaquin Lopez-Contreras Danish Council for Independent Research Sapere Aude, DFF Starting Grant 2014 Andres Joaquin Lopez-Contreras European Research Council ERC-2015-STG-679068 Andres Joaquin Lopez-Contreras Danish National Research Foundation DNRF115 Andres Joaquin Lopez-Contreras The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. Unrepaired DNA damage during embryonic development can be potentially inherited by a large population of cells. However, the quality control mechanisms that minimize the contribution of damaged cells to developing embryos remain poorly understood. Here, we uncovered an ATR- and CHK1-mediated transcriptional response to replication stress (RS) in mouse embryonic stem cells (ESCs) that induces genes expressed in totipotent two-cell (2C) stage embryos and 2C-like cells. This response is mediated by Dux, a multicopy retrogene defining the cleavage-specific transcriptional program in placental mammals. In response to RS, DUX triggers the transcription of 2C-like markers such as murine endogenous retrovirus-like elements (MERVL) and Zscan4. This response can also be elicited by ETAA1-mediated ATR activation in the absence of RS. ATR-mediated activation of DUX requires GRSF1-dependent post-transcriptional regulation of Dux mRNA. Strikingly, activation of ATR expands ESCs fate potential by extending their contribution to both embryonic and extra-embryonic tissues. These findings define a novel ATR dependent pathway involved in maintaining genome stability in developing embryos by controlling ESCs fate in response to RS. Sí

Pluripotent stem cell (PSC) cultures are subjected to selective pressures that can result in acquisition and expansion of recurrent genetic abnormalities at any time. These recurrent abnormalities enhance the variant cells harboring them with a competitive advantage over wild-type cells. Variant cells can eventually supplant wild-type cells entirely and become fixed in culture. Such variants can impact the efficacy of PSCs in research and clinical applications. Therefore, routine genomic characterization is required for reliable and effective use of PSCs. In this article we describe the capabilities and limitations of several assays commonly used for assessing PSC genomic stability. Based on this analysis, we provide a recommendation for integrating assays into a comprehensive testing regimen that maximizes coverage while minimizing cost. © 2020 by John Wiley & Sons, Inc.

Replication-coupled (RC) nucleosome assembly is an essential process in eukaryotic cells in order to maintain chromatin structure during DNA replication. The deposition of newly synthesized H3/H4 histones during DNA replication is facilitated by specialized histone chaperones. Although the contribution of these histone chaperones to genomic stability has been thoroughly investigated, their effect on replisome progression is much less understood. By exploiting a time-lapse microscopy system for monitoring DNA replication in individual live cells, we examined how mutations in key histone chaperones includingCAC1,RTT106,RTT109andASF1, affect replication fork progression. Our experiments revealed that mutations inCAC1orRTT106that directly deposit histones on the DNA, slowdown replication fork progression. In contrast, analysis of cells mutated in the intermediaryASF1orRTT109histone chaperones revealed that replisome progression is not affected. We found that mutations in histone chaperones includingASF1andRTT109lead to extended G2/M duration, elevated number of RPA foci and in some cases, increased spontaneous mutation rate. Our research suggests that histone chaperones have distinct roles in enabling high replisome progression and maintaining genome stability during cell cycle progression.Author SummaryHistone chaperones (HC) play key roles in maintaining the chromatin structure during DNA replication in eukaryotic cells. Despite extensive studies on HCs, little is known regarding their importance for replication fork progression during S-phase. Here, we utilized a live-cell imaging approach to measure the progression rates of single replication forks in individual yeast cells mutated in key histone chaperones. Using this approach, we show that mutations inCAC1orRTT106HCs that directly deposit histones on the DNA lead to slowdown of replication fork progression. In contrast, mutations inASF1orRTT109HCs that transfers H3/H4 toCAC1orRTT106, do not affect replisome progression but lead to post replication defects. Our results reveal distinct functions of HCs in replication fork progression and maintaining genome stability.