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Genomic instability and epigenetic alterations are some of the prominent factors affecting aging. Age-related heterochromatin loss and decreased whole-genome DNA methylation are associated with abnormal gene expression, leading to diseases and genomic instability. Modulation of these epigenetic changes is crucial for preserving genomic integrity and controlling cellular identity is important for slowing the aging process. Numerous studies have shown that caloric restriction is the gold standard for promoting longevity and healthy aging in various species ranging from rodents to primates. It can be inferred that delaying of aging through the main effector such as calorie restriction is involved in cellular identity and epigenetic modification. Thus, an understanding of aging through calorie restriction may seek a more in-depth understanding. In this review, we discuss how caloric restriction promotes longevity and healthy aging through genomic stability and epigenetic alterations. We have also highlighted how the effectors of caloric restriction are involved in modulating the chromatin-based barriers. [ABSTRACT FROM AUTHOR]

Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) hold great potential in regenerative medicine. These cells can be expanded indefinitely in theory and are able to differentiate into different types of cells for cell therapies, drug screening, and basic biology studies. The reliable and effective propagation of hESCs and hiPSCs is important for their downstream applications. Basic fibroblast growth factor (bFGF) is critical to hESCs and hiPSCs for maintaining their pluripotency. Plant-produced growth factors are safe to use without potential contamination of infectious viruses and are less expensive to produce. In this study, we used rice cell-made basic fibroblast growth factor (RbFGF) to propagate hESCs and hiPSCs for at least eight passages. Both hESCs and hiPSCs cultured with RbFGF not only maintained the morphology but also the specific expression (OCT4, SSEA4, SOX2, and TRA-1-60) of PSCs, similar to those cultured with the commercial Escherichia coli-produced bFGF. Furthermore, both gene chip-based PluriTest and TaqMan hPSC Scorecard pluripotency analysis demonstrated the pluripotent expression profile of the hESCs cultured with RbFGF. In vitro trilineage assays further showed that these hESCs and hiPSCs cultured on RbFGF were capable of giving rise to cell derivatives of ectoderm, mesoderm, and endoderm, further demonstrating their pluripotency. Finally, chromosome stability was also maintained in hESCs cultured with RbFGF as demonstrated by normal karyotypes. This study suggests broad applications for plant-made growth factors in stem cell culture and regenerative medicine. [ABSTRACT FROM AUTHOR]

ABSTRACT Furfural is a major inhibitor found in lignocellulosic hydrolysate, a promising feedstock for the biofermentation industry. In this study, we aimed to investigate the potential impact of this furan-derived chemical on yeast genome integrity and phenotypic evolution by using genetic screening systems and high-throughput analyses. Our results showed that the rates of aneuploidy, chromosomal rearrangements (including large deletions and duplications), and loss of heterozygosity (LOH) increased by 50-fold, 23-fold, and 4-fold, respectively, when yeast cells were cultured in medium containing a nonlethal dose of furfural (0.6 g/L). We observed significantly different ratios of genetic events between untreated and furfural-exposed cells, indicating that furfural exposure induced a unique pattern of genomic instability. Furfural exposure also increased the proportion of CG-to-TA and CG-to-AT base substitutions among point mutations, which was correlated with DNA oxidative damage. Interestingly, although monosomy of chromosomes often results in the slower growth of yeast under spontaneous conditions, we found that monosomic chromosome IX contributed to the enhanced furfural tolerance. Additionally, terminal LOH events on the right arm of chromosome IV, which led to homozygosity of the SSD1 allele, were associated with furfural resistance. This study sheds light on the mechanisms underlying the influence of furfural on yeast genome integrity and adaptability evolution. IMPORTANCE Industrial microorganisms are often exposed to multiple environmental stressors and inhibitors during their application. This study demonstrates that nonlethal concentrations of furfural in the culture medium can significantly induce genome instability in the yeast Saccharomyces cerevisiae. Notably, furfural-exposed yeast cells displayed frequent chromosome aberrations, indicating the potent teratogenicity of this inhibitor. We identified specific genomic alterations, including monosomic chromosome IX and loss of heterozygosity of the right arm of chromosome IV, that confer furfural tolerance to a diploid S. cerevisiae strain. These findings enhance our understanding of how microorganisms evolve and adapt to stressful environments and offer insights for developing strategies to improve their performance in industrial applications.

The interest in Robertsonian fusion chromosomes (Rb fusions), sometimes referred to as Robertsonian translocations, derives from their impact on mammalian karyotype evolution, as well from their influence on fertility and disease. The formation of a Rb chromosome necessitates the occurrence of double strand breaks in the pericentromeric regions of two chromosomes in the satellite DNA (satDNA) sequences. Here, we report on the fine-scale molecular analysis of the centromeric satDNA families in the Rb(1;29) translocation of domestic cattle and six antelope species of the subfamily Bovinae. We do so from two perspectives: its occurrence as a chromosomal abnormality in cattle and, secondly, as a fixed evolutionarily rearrangement in spiral-horned antelope (Tragelaphini). By analysing the reorganization of satDNAs in the centromeric regions of translocated chromosomes, we show that Rb fusions are multistep, complex rearrangements which entail the precise elimination and reorganization of specific (peri)centromeric satDNA sequences. Importantly, these structural changes do not influence the centromeric activity of the satellite DNAs that provide segregation stability to the translocated chromosome. Our results suggest a common mechanism for Rb fusions in these bovids and, more widely, for mammals in general. [ABSTRACT FROM AUTHOR]

Fanconi anemia (FA) is a rare genetic disease characterized by increased risk for bone marrow failure and cancer. The FA proteins function together to repair damaged DNA. A central step in the activation of the FA pathway is the monoubiquitination of the FANCD2 and FANCI proteins, which occurs upon exposure to DNA-damaging agents and during the S phase of the cell cycle. The regulatory mechanisms governing S-phase monoubiquitination, in particular, are poorly understood. In this study, we have identified a cyclin-dependent kinase (CDK) regulatory phosphosite (S592) proximal to the site of FANCD2 monoubiquitination. FANCD2 S592 phosphorylation was detected by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and by immunoblotting with an S592 phospho-specific antibody. Mutation of S592 leads to abrogated monoubiquitination of FANCD2 during the S phase. Furthermore, FA-D2 (FANCD2-/-) patient cells expressing S592 mutants display reduced proliferation under conditions of replication stress and increased mitotic aberrations, including micronuclei and multinucleated cells. Our findings describe a novel cell cycle-specific regulatory mechanism for the FANCD2 protein that promotes mitotic fidelity. [ABSTRACT FROM AUTHOR]

Immunodeficiency, centromeric instability, and facial anomalies (ICF) syndrome is characterized by frequent appearance of multiradial chromosomes, which are distinctive chromosome fusions that occur at hypomethylated pericentromeric regions comprising repetitive sequences, in activated lymphocytes. The syndrome is caused by mutations in DNMT3B, ZBTB24, CDCA7, or HELLS. De novo DNA methylation is likely defective in patients with ICF syndrome harboring mutations in DNMT3B, whereas accumulating evidence suggests that replication‐uncoupled maintenance DNA methylation of late‐replicating regions is impaired in patients with ICF syndrome harboring mutations in ZBTB24, CDCA7, or HELLS. ZBTB24 is a transcriptional activator of CDCA7, and CDCA7 and HELLS compose a chromatin remodeling complex and are involved in the maintenance DNA methylation through an interaction with UHRF1 in a feed‐forward manner. Furthermore, our recent studies possibly provided the missing link between DNA hypomethylation and the formation of the abnormal chromosomes; it could occur via aberrant transcription from the hypomethylated regions, followed by pathological R‐loop formation. The homologous‐recombination dominant condition caused by a defect in nonhomologous end joining observed in several types of ICF syndrome could facilitate the formation of multiradial chromosomes. Here, the latest knowledge regarding maintenance DNA methylation and chromosome stability provided by those studies is reviewed. [ABSTRACT FROM AUTHOR]

The disease-associated nuclease–helicase DNA2 has been implicated in DNA end-resection during DNA double-strand break repair, Okazaki fragment processing, and the recovery of stalled DNA replication forks (RFs). Its role in Okazaki fragment processing has been proposed to explain why DNA2 is indispensable for cell survival across organisms. Unexpectedly, we found that DNA2 has an essential role in suppressing homologous recombination (HR)-dependent replication restart at stalled RFs. In the absence of DNA2-mediated RF recovery, excessive HR-restart of stalled RFs results in toxic levels of abortive recombination intermediates that lead to DNA damage-checkpoint activation and terminal cell-cycle arrest. While HR proteins protect and restart stalled RFs to promote faithful genome replication, these findings show how HR-dependent replication restart is actively constrained by DNA2 to ensure cell survival. These new insights disambiguate the effects of DNA2 dysfunction on cell survival, and provide a framework to rationalize the association of DNA2 with cancer and the primordial dwarfism disorder Seckel syndrome based on its role in RF recovery. [ABSTRACT FROM AUTHOR]

Plants have the salient biological property of totipotency, i.e., the capacity to regenerate a whole plant from virtually any kind of fully differentiated somatic cells after a process of dedifferentiation. This property has been well-documented by successful plant regeneration from tissue cultures of diverse plant species. However, the accumulation of somaclonal variation, especially karyotype alteration, during the tissue culture process compromises cell totipotency. In this respect, Chinese ginseng (Panax ginseng C. A. Mey.) is an exception in that it shows little decline in cell totipotency accompanied by remarkable chromosomal stability even after prolonged tissue cultures. However, it remains unclear whether chromosomal level stability necessarily couples with molecular genetic stability at the nucleotide sequence level, given that the two types of stabilities are generated by largely distinct mechanisms. Here, we addressed this issue by genome-wide comparisons at the single-base resolution of long-term tissue culture-regenerated P. ginseng plants. We identified abundant single nucleotide polymorphisms (SNPs) that have accumulated in cultured ginseng callus and are retained in the process of plant regeneration. These SNPs did not occur at random but showed differences among chromosomes and biased regional aggregation along a given chromosome. In addition, our results demonstrate that, compared with the overall genes, genes related to processes of cell totipotency and chromosomal stability possess lower mutation rates at both coding and flanking regions. In addition, collectively, the mutated genes exhibited higher expression levels than non-mutated genes and are significantly enriched in fundamental biological processes, including cellular component organization, development, and reproduction. These attributes suggest that the precipitated molecular level genetic variations during the process of regeneration in P. ginseng are likely under selection to fortify normal development. As such, they likely did not undermine chromosomal stability and totipotency of the long-term ginseng cultures.

Chromatin integrity is essential for cellular homeostasis. Polycomb group proteins modulate chromatin states and transcriptionally repress developmental genes to maintain cell identity. They also repress repetitive sequences such as major satellites and constitute an alternative state of pericentromeric constitutive heterochromatin at paternal chromosomes (pat‐PCH) in mouse pre‐implantation embryos. Remarkably, pat‐PCH contains the histone H3.3 variant, which is absent from canonical PCH at maternal chromosomes, which is marked by histone H3 lysine 9 trimethylation (H3K9me3), HP1, and ATRX proteins. Here, we show that SUMO2‐modified CBX2‐containing Polycomb Repressive Complex 1 (PRC1) recruits the H3.3‐specific chaperone DAXX to pat‐PCH, enabling H3.3 incorporation at these loci. Deficiency of Daxx or PRC1 components Ring1 and Rnf2 abrogates H3.3 incorporation, induces chromatin decompaction and breakage at PCH of exclusively paternal chromosomes, and causes their mis‐segregation. Complementation assays show that DAXX‐mediated H3.3 deposition is required for chromosome stability in early embryos. DAXX also regulates repression of PRC1 target genes during oogenesis and early embryogenesis. The study identifies a novel critical role for Polycomb in ensuring heterochromatin integrity and chromosome stability in mouse early development. Synopsis: Formation of pericentromeric heterochromatin (PCH) in early mouse embryos involves selective incorporation of the histone variant H3.3. Polycomb repressive complex 1 (PRC1) facilitates this by recruitment of H3.3 chaperone/remodeler DAXX‐ATRX in a SUMOylation‐dependent manner. Histone chaperone DAXX controls H3.3 deposition into pericentromeric heterochromatin exclusively at paternal chromosomes of early mouse embryos.Canonical PRC1, but not PRC2, is required for recruitment of DAXX and H3.3 deposition at paternal PCH.SUMOylation of the PRC1 component CBX2 mediates interaction between CBX2 and DAXX.DAXX contributes to PRC1‐dependent gene repression in oocytes. [ABSTRACT FROM AUTHOR]

The SKP1, CUL1, F-box protein (SCF) complex encompasses a group of 69 SCF E3 ubiquitin ligase complexes that primarily modify protein substrates with poly-ubiquitin chains to target them for proteasomal degradation. These SCF complexes are distinguishable by variable F-box proteins, which determine substrate specificity. Although the function(s) of each individual SCF complex remain largely unknown, those that have been characterized regulate a wide array of cellular processes, including gene transcription and the cell cycle. In this regard, the SCF complex regulates transcription factors that modulate cell signaling and ensures timely degradation of primary cell cycle regulators for accurate replication and segregation of genetic material. SCF complex members are aberrantly expressed in a myriad of cancer types, with altered expression or function of the invariable core SCF components expected to have a greater impact on cancer pathogenesis than that of the F-box proteins. Accordingly, this review describes the normal roles that various SCF complexes have in maintaining genome stability before discussing the impact that aberrant SCF complex expression and/or function have on cancer pathogenesis. Further characterization of the SCF complex functions is essential to identify and develop therapeutic approaches to exploit aberrant SCF complex expression and function.

DNA double-strand breaks (DSBs) disrupt the structural integrity of chromosomes. Proper DSB repair pathway choice is critical to avoid the type of gross chromosomal rearrangements that characterize cancer cells. Recent findings reveal S-fatty acylation and membrane anchorage of Rap1-interacting factor 1 (Rif1) as a mechanism providing spatial control over DSB repair pathway choice.

The conserved nuclease-helicase DNA2 has been linked to mitochondrial myopathy, Seckel syndrome, and cancer. Across species, the protein is indispensable for cell proliferation. On the molecular level, DNA2 has been implicated in DNA double-strand break (DSB) repair, checkpoint activation, Okazaki fragment processing (OFP), and telomere homeostasis. More recently, a critical contribution of DNA2 to the replication stress response and recovery of stalled DNA replication forks (RFs) has emerged. Here, we review the available functional and phenotypic data and propose that the major cellular defects associated with DNA2 dysfunction, and the links that exist with human disease, can be rationalized through the fundamental importance of DNA2-dependent RF recovery to genome duplication. Being a crucial player at stalled RFs, DNA2 is a promising target for anti-cancer therapy aimed at eliminating cancer cells by replication-stress overload.

Maintenance of genome stability is a crucial cellular function for normal mammalian development and physiology. However, despite the general relevance of this process, genome stability alteration due to genetic or non-genetic conditions has a particularly profound impact on the developing cerebral cortex. In this review, we will analyze the main pathways involved in maintenance of genome stability, the consequences of their alterations with regard to central nervous system development, as well as the possible molecular and cellular basis of this specificity. [ABSTRACT FROM AUTHOR]

Key message: Standard pathways involved in the regulation of telomere stability do not contribute to gradual telomere elongation observed in the course of A. thaliana calli propagation.Abstract: Genetic and epigenetic changes accompanying the culturing of plant cells have frequently been reported. Here we aimed to characterize the telomere homeostasis during long term callus propagation. While in Arabidopsis thaliana calli gradual telomere elongation was observed, telomeres were stable in Nicotiana tabacum and N. sylvestris cultures. Telomere elongation during callus propagation is thus not a general feature of plant cells. The long telomere phenotype in Arabidopsis calli was correlated neither with changes in telomerase activity nor with activation of alternative mechanisms of telomere elongation. The dynamics of telomere length changes was maintained in mutant calli with loss of function of important epigenetic modifiers but compromised in the presence of epigenetically active drug zebularine. To examine whether the cell culture-induced disruption of telomere homeostasis is associated with the modulated structure of chromosome ends, epigenetic properties of telomere chromatin were analysed. Albeit distinct changes in epigenetic modifications of telomere histones were observed, these were broadly stochastic. Our results show that contrary to animal cells, the structure and function of plant telomeres is not determined significantly by the epigenetic character of telomere chromatin. Set of differentially transcribed genes was identified in calli, but considering the known telomere- or telomerase-related functions of respective proteins, none of these changes per se was apparently related to the elongated telomere phenotype. Based on our data, we propose that the disruption in telomere homeostasis in Arabidopsis calli arises from the interplay of multiple factors, as a part of reprogramming of plant cells to long-term culture conditions. [ABSTRACT FROM AUTHOR]

Genomic stability is maintained by the concerted actions of numerous protein complexes that participate in chromosomal duplication, repair, and segregation. The Smc5/6 complex is an essential multi-subunit complex crucial for repair of DNA double-strand breaks. Two of its subunits, Nse1 and Nse3, are homologous to the RING-MAGE complexes recently described in human cells. We investigated the contribution of the budding yeast Nse1 RING-domain by isolating a mutant nse1-103 bearing substitutions in conserved Zinc-coordinating residues of the RING-domain that is hypersensitive to genotoxic stress and temperature. The nse1-103 mutant protein was defective in interaction with Nse3 and other Smc5/6 complex subunits, Nse4 and Smc5. Chromosome loss was enhanced, accompanied by a delay in the completion of replication and a modest defect in sister chromatid cohesion, in nse1-103. The nse1-103 mutant was synthetic sick with rrm3∆ (defective in fork passage through pause sites), this defect was rescued by inactivation of Tof1, a subunit of the fork protection complex that enforces pausing. The temperature sensitivity of nse1-103 was partially suppressed by deletion of MPH1, encoding a DNA-helicase. Homology modeling of the structure of the budding yeast Nse1-Nse3 heterodimer based on the human Nse1-MAGEG1 structure suggests a similar organization and indicates that perturbation of the Zn-coordinating cluster has the potential to allosterically alter structural elements at the Nse1/Nse3 interaction interface that may abrogate their association. Our findings demonstrate that the budding yeast Nse1 RING-domain organization is important for interaction with Nse3, which is crucial for completion of chromosomal replication, cohesion, and maintenance of chromosome stability. [ABSTRACT FROM AUTHOR]

This study was aimed to investigate the stability of chromosomes during meiosis in autopolyploid and allopolyploid maize, as well as to determine an association of chromosomes between maize (Zea mays ssp. mays Linnaeus, 1753) and Z. perennis (Hitchcock, 1922) Reeves & Mangelsdor, 1942, by producing a series of autopolyploid and allopolyploid maize hybrids. The intra-genomic and inter-genomic meiotic pairings in these polyploids were quantified and compared using dual-color genomic in-situ hybridization. The results demonstrated higher level of chromosome stability in allopolyploid maize during meiosis as compared to autopolyploid maize. In addition, the meiotic behavior of Z. perennis was relatively more stable as compared to the allopolyploid maize. Moreover, ten chromosomes of "A" subgenome in maize were homologous to twenty chromosomes of Z. perennis genome with a higher pairing frequency and little evolutionary differentiation. At the same time, little evolutionary differentiation has been shown by chromosomes of "A" subgenome in maize, while chromosomes of "B" subgenome, had a lower pairing frequency and higher evolutionary differentiation. Furthermore, 5IM + 5IIPP + 5IIIMPP and 5IIMM + 5IIPP + 5IVMMPP were observed in allotriploids and allotetraploids respectively, whereas homoeologous chromosomes were found between the "A" and "B" genome of maize and Z. perennis. [ABSTRACT FROM AUTHOR]

Phenylpropanoid glycosides (PPG), like other phenolic compounds, are a powerful antioxidants and the Verbascoside (VB) is one of the most active of them. A previous study, by using in vitro exposure of blood human lymphocytes to Verbascoside, reported a significant increasings of chromosome fragility compared to control. In the present study, four homogeneous groups of rabbits were used to test in vivo the VB and/or Lycopene (LP) by feeding the animals without VB and LP (control), in presence of VB or/and LP for 80 days. Lymphocyte cell cultures were performed in three different times: 0, 40 and 80 days of the experiment and the cytogenetic tests that we used [CA-test (Chromosome Abnormalities in terms of chromosome and chromatid breaks) and Sister Chromatid Exchange (SCE-test)] have revealed no mutagenic effects on chromosomes. Indeed, mean values/cell of CA and SCE decreased during the experiment with some difference among and within groups, with significant decreasing value only for some group. The study shows clear evidence that diets rich in Verbascoside (and/or Lycopene) do not originate any mutagenic activity, resulting no cytotoxic for the animals and, suggesting a possible their use in both animal and human diets. [ABSTRACT FROM AUTHOR]

ABSTRACT The human malaria parasite Plasmodium falciparum replicates within circulating red blood cells, where it is subjected to conditions that frequently cause DNA damage. The repair of DNA double-stranded breaks (DSBs) is thought to rely almost exclusively on homologous recombination (HR), due to a lack of efficient nonhomologous end joining. However, given that the parasite is haploid during this stage of its life cycle, the mechanisms involved in maintaining genome stability are poorly understood. Of particular interest are the subtelomeric regions of the chromosomes, which contain the majority of the multicopy variant antigen-encoding genes responsible for virulence and disease severity. Here, we show that parasites utilize a competitive balance between de novo telomere addition, also called “telomere healing,” and HR to stabilize chromosome ends. Products of both repair pathways were observed in response to DSBs that occurred spontaneously during routine in vitro culture or resulted from experimentally induced DSBs, demonstrating that both pathways are active in repairing DSBs within subtelomeric regions and that the pathway utilized was determined by the DNA sequences immediately surrounding the break. In combination, these two repair pathways enable parasites to efficiently maintain chromosome stability while also contributing to the generation of genetic diversity. IMPORTANCE Malaria is a major global health threat, causing approximately 430,000 deaths annually. This mosquito-transmitted disease is caused by Plasmodium parasites, with infection with the species Plasmodium falciparum being the most lethal. Mechanisms underlying DNA repair and maintenance of genome integrity in P. falciparum are not well understood and represent a gap in our understanding of how parasites survive the hostile environment of their vertebrate and insect hosts. Our work examines DNA repair in real time by using single-molecule real-time (SMRT) sequencing focused on the subtelomeric regions of the genome that harbor the multicopy gene families important for virulence and the maintenance of infection. We show that parasites utilize two competing molecular mechanisms to repair double-strand breaks, homologous recombination and de novo telomere addition, with the pathway used being determined by the surrounding DNA sequence. In combination, these two pathways balance the need to maintain genome stability with the selective advantage of generating antigenic diversity.

The exploitation of synthetic lethality by small-molecule targeting of pathways that maintain genomic stability is an attractive chemotherapeutic approach. The Ctf4/AND-1 protein hub, which links DNA replication, repair, and chromosome segregation, represents a novel target for the synthetic lethality approach. Herein, we report the design, optimization, and validation of double-click stapled peptides encoding the Ctf4-interacting peptide (CIP) of the replicative helicase subunit Sld5. By screening stapling positions in the Sld5 CIP, we identified an unorthodox i,i+6 stapled peptide with improved, submicromolar binding to Ctf4. The mode of interaction with Ctf4 was confirmed by a crystal structure of the stapled Sld5 peptide bound to Ctf4. The stapled Sld5 peptide was able to displace the Ctf4 partner DNA polymerase α from the replisome in yeast extracts. Our study provides proof-of-principle evidence for the development of small-molecule inhibitors of the human CTF4 orthologue AND-1. [ABSTRACT FROM AUTHOR]

Replication of linear chromosomes is facilitated by firing of multiple replication origins that ensures timely duplication of the entire chromosome. The Smc5/6 complex is thought to play an important role in replication by its involvement in the restart of collapsed replication forks. Here, we present genetic evidence for functional interaction between replication origin distribution and two subunits of the Smc5/6 complex, Smc6 and Mms21, as well as Top1. An artificial chromosome that has a long arm having low origin density (5ori∆YAC) is relatively unstable compared to the YAC having normal origin distribution in wild-type cells, but is partially stabilized in smc6-56 and top1∆ mutants. While a SUMO-ligase-deficient mutant of Mms21 does not affect stability of the 5ori∆YAC by itself, in combination with top1∆, the 5ori∆YAC is destabilized as evidenced by increased chromosome loss frequency in the mms21∆sl top1∆ double mutant. Likewise, the smc6-56 top1∆ double mutant also exhibits enhanced destabilization of the 5ori∆YAC compared to either single mutant. Such an increase in chromosome loss is not observed for a similar YAC that retains the original replication origins and normal origin distribution on the long arm, in either double mutant having the mms21∆sl or smc6-56 mutations in combination with top1∆. Our findings reveal a requirement for the Smc5/6 complex, including Mms21/Nse2 mediated sumoylation, and topoisomerase-1 (Top1), for maintaining stability of a chromosome having low origin density and suggest a functional cooperation between the Smc5/6 complex and Top1 in maintenance of topologically challenged chromosomes prone to replication fork collapse or accumulation of torsional stress. [ABSTRACT FROM AUTHOR]

Genetic information in cells is encrypted in DNA molecules forming chromosomes of varying sizes. Accurate replication and partitioning of chromosomes in the crowded cellular milieu is a complex process involving duplication, folding and movement. Longer chromosomes may be more susceptible to mis-segregation or DNA damage and there may exist specialized physiological mechanisms preventing this. Here, we present genetic evidence for such a mechanism which depends on Mms21/Nse2 mediated sumoylation and topoisomerase-1 (Top1) for maintaining stability of longer chromosomes. While mutations inactivating Top1 or the SUMO ligase activity of Mms21 ( mms21sl) individually destabilized yeast artificial chromosomes (YACs) to a modest extent, the mms21sl top1 double mutant exhibited a synthetic-sick phenotype, and showed preferential destabilization of the longer chromosome relative to shorter chromosomes. In contrast, an smc6- 56 top1 mutant defective in Smc6, another subunit of the Smc5/6 complex, of which Mms21 is a component, did not show such a preferential enhancement in frequency of loss of the longer YAC, indicating that this defect may be specific to the deficiency in SUMO ligase activity of Mms21 in the mms21sl top1 mutants. In addition, mms21sl top1 double mutants harboring a longer fusion derivative of natural yeast chromosomes IV and XII displayed reduced viability, consistent with enhanced chromosome instability, relative to single mutants or the double mutant having the natural (shorter) non-fused chromosomes. Our findings reveal a functional interplay between Mms21 and Top1 in maintenance of longer chromosomes, and suggest that lack of sumoylation of Mms21 targets coupled with Top1 deficiency is a crucial requirement for accurate inheritance of longer chromosomes. [ABSTRACT FROM AUTHOR]

Furfural is a major inhibitor found in lignocellulosic hydrolysate, a promising feedstock for the biofermentation industry. In this study, we aimed to investigate the potential impact of this furan-derived chemical on yeast genome integrity and phenotypic evolution by using genetic screening systems and high-throughput analyses. Our results showed that the rates of aneuploidy, chromosomal rearrangements (including large deletions and duplications), and loss of heterozygosity (LOH) increased by 50-fold, 23-fold, and 4-fold, respectively, when yeast cells were cultured in medium containing a nonlethal dose of furfural (0.6 g/L). We observed significantly different ratios of genetic events between untreated and furfural-exposed cells, indicating that furfural exposure induced a unique pattern of genomic instability. Furfural exposure also increased the proportion of CG-to-TA and CG-to-AT base substitutions among point mutations, which was correlated with DNA oxidative damage. Interestingly, although monosomy of chromosomes often results in the slower growth of yeast under spontaneous conditions, we found that monosomic chromosome IX contributed to the enhanced furfural tolerance. Additionally, terminal LOH events on the right arm of chromosome IV, which led to homozygosity of the SSD1 allele, were associated with furfural resistance. This study sheds light on the mechanisms underlying the influence of furfural on yeast genome integrity and adaptability evolution. IMPORTANCE Industrial microorganisms are often exposed to multiple environmental stressors and inhibitors during their application. This study demonstrates that nonlethal concentrations of furfural in the culture medium can significantly induce genome instability in the yeast Saccharomyces cerevisiae. Notably, furfural-exposed yeast cells displayed frequent chromosome aberrations, indicating the potent teratogenicity of this inhibitor. We identified specific genomic alterations, including monosomic chromosome IX and loss of heterozygosity of the right arm of chromosome IV, that confer furfural tolerance to a diploid S. cerevisiae strain. These findings enhance our understanding of how microorganisms evolve and adapt to stressful environments and offer insights for developing strategies to improve their performance in industrial applications., Competing Interests: The authors declare no conflict of interest.

DNA double-strand breaks (DSBs) disrupt the structural integrity of chromosomes. Proper DSB repair pathway choice is critical to avoid the type of gross chromosomal rearrangements that characterize cancer cells. Recent findings reveal S-fatty acylation and membrane anchorage of Rap1-interacting factor 1 (Rif1) as a mechanism providing spatial control over DSB repair pathway choice. [ABSTRACT FROM AUTHOR]

Telomerase is essential for regulating telomeres, and its activation is a critical step in cellular immortalization and tumorigenesis. The transcriptional activation of human telomerase reverse transcriptase (hTERT) is critical for telomerase expression. Although several transcriptional activators have been identified, factors responsible for enhancing the hTERT promoter remain to be fully elucidated. In the present study, the role of glioma tumor-suppressor candidate region gene 2 (GLTSCR2) in telomerase regulation was analyzed. A doxycyclin-inducible green fluorescent protein (GFP)-tagged GLTSCR2-expressing adenovirus (Ad-GLT/GFP) was used for the transduction of SK-Hep-1 and T98G cancer cells, and normal human umbilical vein endothelial cells. Changes in telomerase activity using telomere repeat amplification protocol assay were assessed, and the gene expression levels of hTERT were then examined. To investigate chromosome instability and senescence, Giemsa and β-galactosidase staining was performed. The results revealed that overexpression of GLTSCR2 significantly increased telomerase activity in the cancer and normal cell lines. This increase was consistent with increases in the protein and mRNA expression levels of hTERT. In luciferase assays, the hTERT promoter was activated by GLTSCR2. Knockdown of GLTSCR2 led to the downregulation of telomerase activity, abnormal nuclear morphology as a marker of chromosome instability, significant suppression of growth rate, alterations in cellular morphology and, eventually, cellular senescence. Taken together, the results of the present study suggested that GLTSCR2 is crucially involved in the positive regulation of telomerase and chromosome stability. [ABSTRACT FROM AUTHOR]

Gene-environment interactions can perturb the epigenome, triggering network alterations that participate in cancer pathogenesis. Integrating epigenomics, transcriptomics, and metabolic analyses with functional perturbation, we show that the tumor suppressor p53 preserves genomic integrity by empowering adequate levels of the universal methyl donor S-adenosylmethionine (SAM). In p53-deficient cells, perturbation of DNA methylation promotes derepression of heterochromatin, massive loss of histone H3-lysine 9 methylation, and consequent upregulation of satellite RNAs that triggers R-loop-associated replication stress and chromosomal aberrations. In p53-deficient cells, the inadequate SAM level underlies the inability to respond to perturbation because exogenous reintroduction of SAM represses satellite elements and restores the ability to cope with stress. Mechanistically, p53 transcriptionally controls genes involved in one-carbon metabolism, including Slc43a2, the methionine uptake transporter that is critical for SAM synthesis. Supported by clinical data, our findings shed light on the role of p53-mediated metabolism in preventing unscheduled R-loop-associated genomic instability. [Display omitted] • p53 inactivation reduces the level of S-adenosylmethionine (SAM) • p53 preserves stressed constitutive heterochromatin • p53 prevents aberrant satellite transcription and R-loop-dependent genomic instability • Restoration of SAM reinstates genomic integrity in p53 deficiency Panatta et al. demonstrate that cells lacking functional p53 when exposed to stress undergo genomic instability as a consequence of inability to preserve the constitutive heterochromatin status. p53 ensures adequate level of S-adenosylmethionine, which instates persistence of histone methylation and prevents loss of genomic integrity. [ABSTRACT FROM AUTHOR]

Targeting cardiomyocyte plasticity has emerged as a new strategy for promoting heart repair after myocardial infarction. However, the precise mechanistic network underlying heart regeneration is not completely understood. As noncoding RNAs, circular RNAs (circRNAs) play essential roles in regulating cardiac physiology and pathology. The present study aimed to investigate the potential roles of circMdc1 in cardiac repair after injury and elucidate its underlying mechanisms. Here, we identified that circMdc1 levels were upregulated in postnatal mouse hearts but downregulated in the regenerative myocardium. The expression of circMdc1 in cardiomyocytes is sensitive to oxidative stress, which was attenuated by N-acetyl-cysteine. Enforced circMdc1 expression inhibited cardiomyocyte proliferation, while circMdc1 silencing led to cardiomyocyte cell cycle re-entry. In vivo , the cardiac-specific adeno-associated virus-mediated knockdown of circMdc1 promoted cardiac regeneration and heart repair accompanied by improved heart function. Conversely, circMdc1 overexpression blunted the regenerative capacity of neonatal hearts after apex resection. Moreover, circMdc1 was able to block the translation of its host gene Mdc1 specifically by binding to PABP, affecting DNA damage and the chromosome stability of cardiomyocytes. Furthermore, overexpression of Mdc1 caused damaged mouse hearts to regenerate and repair after myocardial infarction in vivo. Oxidative stress-sensitive circMdc1 plays an important role in cardiac regeneration and heart repair after injury by regulating DNA damage and chromosome stability in cardiomyocytes by blocking the translation of the host gene Mdc1. [Display omitted] [ABSTRACT FROM AUTHOR]

Diffuse large B cell lymphoma (DLBCL) is the most common form of lymphoma in the United States. DLBCL comprises biologically distinct subtypes including germinal center-like (GCB) and activated-B-cell-like DLBCL (ABC). The most aggressive type, ABC-DLBCL, displays dysregulation of both canonical and noncanonical NF-κB pathway as well as genomic instability. Although, much is known about the tumorigenic roles of the canonical NF-kB pathway, the precise role of the noncanonical NF-kB pathway remains unknown. Here we show that activation of the noncanonical NF-κB pathway regulates chromosome stability, DNA damage response and centrosome duplication in DLBCL. Analysis of 92 DLBCL samples revealed that activation of the noncanonical NF-κB pathway is associated with low levels of DNA damage and centrosome amplification. Inhibiting the noncanonical pathway in lymphoma cells uncovered baseline DNA damage and prevented doxorubicin-induced DNA damage repair. In addition, it triggered centrosome amplification and chromosome instability, indicated by anaphase bridges, multipolar spindles and chromosome missegregation. We determined that the noncanonical NF-κB pathway execute these functions through the regulation of GADD45α and REDD1 in a p53-independent manner, while it collaborates with p53 to regulate cyclin G2 expression. Furthermore, this pathway regulates GADD45α, REDD1 and cyclin G2 through direct binding of NF-κB sites to their promoter region. Overall, these results indicate that the noncanonical NF-κB pathway plays a central role in maintaining genome integrity in DLBCL. Our data suggests that inhibition of the noncanonical NF-kB pathway should be considered as an important component in DLBCL therapeutic approach. [ABSTRACT FROM AUTHOR]

Telomeres are nucleoprotein complexes that cap the ends of all linear chromosomes and function to prevent aberrant repair and end-to-end chromosome fusions. In somatic cells, telomere shortening is a natural part of the aging process as it occurs with each round of cell division. In germ and stem cells, however, the enzyme telomerase synthesizes telomere DNA to counter-balance telomere shortening and help maintain cellular proliferation. Of the primary telomere end-binding proteins, TPP1 has recently emerged as a primary contributor in protecting telomere DNA and in recruiting telomerase to the telomere ends. In this review, we summarize the current knowledge regarding the role of TPP1 in telomere maintenance. [ABSTRACT FROM AUTHOR]

The major mammalian apurinic/apyrimidinic endonuclease Ape1 is a multifunctional protein operating in protection of cells from oxidative stress via its DNA repair, redox, and transcription regulatory activities. The importance of Ape1 has been marked by previous work demonstrating its requirement for viability in mammalian cells. However, beyond a requirement for Ape1-dependent DNA repair activity, deeper molecular mechanisms of the fundamental role of Ape1 in cell survival have not been defined. Here, we report that Ape1 is an essential factor stabilizing telomeric DNA, and its deficiency is associated with telomere dysfunction and segregation defects in immortalized cells maintaining telomeres by either the alternative lengthening of telomeres pathway (U2OS) or telomerase expression (BJ-hTERT), or in normal human fibroblasts (IMR90). Through the expression of Ape1 derivatives with site-specific changes, we found that the DNA repair and N-terminal acetylation domains are required for the Ape1 function at telomeres. Ape1 associates with telomere proteins in U2OS cells, and Ape1 depletion causes dissociation of TRF2 protein from telomeres. Consistent with this effect, we also observed that Ape1 depletion caused telomere shortening in both BJ-hTERT and in HeLa cells. Thus, our study describes a unique and unpredicted role for Ape1 in telomere protection, providing a direct link between base excision DNA repair activities and telomere metabolism. [ABSTRACT FROM AUTHOR]

Abstract: A family of helicases that are important in maintaining genome stability is the iron–sulfur group. Members of this family include DOG-1/FANCJ, RTEL1, XPD and Chl1p/DDX11. In Caenorhabitis elegans, the predicted gene M03C11.2 has orthology to the CHL1 (Chromosome loss 1) gene in Saccharomyces cerevisiae and DDX11 (DEAD/H box polypeptide 11) in humans. In this paper, we show that the chl-1 gene in C. elegans is required for normal development and fertility. Mutants have lineage-independent cell proliferation defects that result in a Stu (sterile uncoordinated) phenotype, characterized by gonadal abnormalities and a reduced number of D motor neurons and seam cells. A chromosome stability defect is present in the germ cells, where an abnormal number of DAPI-staining chromosomes appear in diakinesis. CHL-1 function is required for the integrity of poly-guanine/poly-cytosine DNA in the absence of DOG-1/FANCJ: the loss of CHL-1 alone does not result in the deletion of G-tracts, but it does increase the number of deletions observed in the dog-1; chl-1 double mutant, indicating a role for CHL-1 during replication and repair. In addition, we observed that cohesin defects increased the number of deletions in the absence of DOG-1/FANCJ. Our results demonstrate a role for CHL-1 in cell proliferation and maintaining normal chromosome numbers, and implicate CHL-1 in chromosome stability and repair of unresolved secondary structures during replication. [Copyright &y& Elsevier]