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The compaction of chromatin into mitotic chromosomes is essential for faithful transmission of the genome during cell division. In eukaryotes, chromosome morphogenesis is regulated by the condensin complex, though the exact mechanism used to target condensin to chromatin and initiate condensation is not understood. Here, we reveal that condensin contains an intrinsically disordered region (IDR) that modulates its association with chromatin in early mitosis and exhibits phase separation. We describe DNA-binding motifs within the IDR that, upon deletion, inflict striking defects in chromosome condensation and segregation, ill-timed condensin turnover on chromatin, and cell death. Importantly, we demonstrate that the condensin IDR can impart cell cycle regulatory functions when transferred to other subunits within the complex, indicating its autonomous nature. Collectively, our study unveils the molecular basis for the initiation of chromosome condensation in early mitosis and how this process ultimately promotes genomic stability and faultless cell division. [Display omitted] • The condensin complex is the main regulator of chromosome condensation in mitosis • Condensin contains an essential intrinsically disordered protein region (IDR) • Purified condensin IDR exhibits phase separation and binds DNA substrates • IDR mutations lead to defective chromosome condensation and segregation During mitosis, chromatin is reorganized into compact chromosomes by the highly dynamic condensin complex. Pastic et al. find that condensin possesses a conformationally flexible, disordered region that is essential for its dynamic association with chromatin. Mutations in this region lead to severe defects in chromosome condensation, segregation, and cell proliferation. [ABSTRACT FROM AUTHOR]

Cancer is caused by a complex interaction of factors that interrupt the normal growth and division of cells. At the center of this process is the intricate relationship between DNA damage and the cellular mechanisms responsible for maintaining genomic stability. When DNA damage is not repaired, it can cause genetic mutations that contribute to the initiation and progression of cancer. On the other hand, the DNA damage response system, which involves the phosphorylation of the histone variant H2AX (γH2AX), is crucial in preserving genomic integrity by signaling and facilitating the repair of DNA double-strand breaks. This review provides an explanation of the molecular dynamics of H2AX in the context of DNA damage response. It emphasizes the crucial role of H2AX in recruiting and localizing repair machinery at sites of chromatin damage. The review explains how H2AX phosphorylation, facilitated by the master kinases ATM and ATR, acts as a signal for DNA damage, triggering downstream pathways that govern cell cycle checkpoints, apoptosis, and the cellular fate decision between repair and cell death. The phosphorylation of H2AX is a critical regulatory point, ensuring cell survival by promoting repair or steering cells towards apoptosis in cases of catastrophic genomic damage. Moreover, we explore the therapeutic potential of targeting H2AX in cancer treatment, leveraging its dual function as a biomarker of DNA integrity and a therapeutic target. By delineating the pathways that lead to H2AX phosphorylation and its roles in apoptosis and cell cycle control, we highlight the significance of H2AX as both a prognostic tool and a focal point for therapeutic intervention, offering insights into its utility in enhancing the efficacy of cancer treatments. • The histone variation H2AX is essential to the DNA damage response and offers a potential cancer therapeutic target. • H2AX is a well-known chromatin modification event in DNA damage response and human malignancies. • H2AX phosphorylation (γ-H2AX) is necessary for DNA repair protein assembly at damaged chromatin regions. • Detecting double strand breaks with phosphorylated histone H2AX may help in cancer treatment. [ABSTRACT FROM AUTHOR]

Genomic instability underlies genesis and the development of various types of cancer. During tumorigenesis, cancer initiating cells assume a set of features, which allow them to survive and proliferate. Different mutations and chromosomal alterations promote a selection of the most aggressive cancer clones that worsen the prognosis of the disease. Despite that caspase-2 was described as a protease fulfilling an initiator and an effector function in apoptosis, it has recently been discovered to play an important role in the maintenance of genomic integrity and normal chromosome configuration. This protein is able to stabilize p53 and affect the level of transcription factors, which activates cell response to oxidative stress. Here we focus on the discussion on the mechanism(s) of how caspase-2 regulates genomic stability and decreases tumorigenesis. Tumors in caspase-2 knockout mice grow rapidly and contain more cells with chromosomal aberrations in contrast to wild-type littermates. An accumulation of genomic disturbances, such as supernumerary centrosomes, activates caspase-2 via PIDDosome formation that restricts the proliferation of polyploid cells. Active caspase-2 can cleave Mdm2 and promote the stabilization of p53 level in response to centrosome amplification, which promotes genomic instability and cell neoplastic transformation. Caspase-2 helps to maintain genomic stability, decreasing the level of oxidative stress and DNA damage. Caspase-2 acts as tumor suppressor via its ability to eliminate cells with chromosomal perturbations or after mitotic insults. [ABSTRACT FROM AUTHOR]

Autophagy is a quality control pathway that maintains cellular homeostasis by recycling surplus and dysregulated cell organelles. Identification of selective autophagy receptors demonstrated the existence of pathways that selectively degrade organelles, protein aggregates or pathogens. Interestingly, different types of DNA damage can induce autophagy and autophagy-deficiency leads to genomic instability. Recent studies provided first insights into the pathways that connect autophagy with the DNA damage response. However, the physiological role of autophagy and the identity of its targets after DNA damage remain enigmatic. In this review, we summarize recent literature on the targets of autophagy and mechanisms that lead to its activation after DNA damage, and discuss potential consequences of DNA damage-induced autophagy. [ABSTRACT FROM AUTHOR]

• The comet assay in assessing genomic stability in relation to folate status and human cancer. • Impact of folate deficiency/supplementation on genomic stability in vitro , in rodents and humans. • The impact of MTHFR genotype on folate status and genomic stability in human carcinogenesis. • Adaptation of the comet assay in measuring DNA methylation and impact on diet and epigenetics. Intake of folate (vitamin B 9) is strongly inversely linked with human cancer risk, particularly colon cancer. In general, people with the highest dietary intake of folate or with high blood folate levels are at a reduced risk (approx. 25%) of developing colon cancer. Folate acts in normal cellular metabolism to maintain genomic stability through the provision of nucleotides for DNA replication and DNA repair and by regulating DNA methylation and gene expression. Folate deficiency can accelerate carcinogenesis by inducing misincorporation of uracil into DNA, by increasing DNA strand breakage, by inhibiting DNA base excision repair capacity and by inducing DNA hypomethylation and consequently aberrant gene and protein expression. Conversely, increasing folate intake may improve genomic stability. This review describes key applications of single cell gel electrophoresis (the comet assay) in assessing genomic instability (misincorporated uracil, DNA single strand breakage and DNA repair capacity) in response to folate status (deficient or supplemented) in human cells in vitro , in rodent models and in human case-control and intervention studies. It highlights an adaptation of the SCGE comet assay for measuring genome-wide and gene-specific DNA methylation in human cells and colon tissue. [ABSTRACT FROM AUTHOR]

Highlights • NME1 depletion down-regulates NHEJ repair of DSBs. • NME1 is recruited to DNA damage sites in a Ku-XRCC4-dependent manner. • The 3′-5′ exonuclease activity of NME1 is critical for LIG4 recruitment and DNA repair efficiency. Abstract NME1 (also known as NM23-H1) was the first identified tumor metastasis suppressor, which has been reported to link with genomic stability maintenance and cancer. However its underlying mechanisms are still not fully understood. Here we find that NME1 is required for non-homologous end joining (NHEJ) of DNA double-strand breaks (DSBs). Mechanistically, NME1 re-localizes to DNA damage sites in a Ku-XRCC4-dependent manner, and regulates downstream LIG4 recruitment and end joining efficiency. Furthermore, we show that the 3′-5′ exonuclease activity of NME1 is critical for its function in NHEJ. Taken together, our findings identify NME1 as a novel NHEJ factor, and reveal how this metastasis suppressor promotes genome stability. [ABSTRACT FROM AUTHOR]

DNA-dependent protein kinase (DNA-PK), a member of phosphatidylinositol-kinase family, is a key protein in mammalian DNA double-strand break (DSB) repair that helps to maintain genomic integrity. DNA-PK also plays a central role in immune cell development and protects telomerase during cellular aging. Epigenetic deregulation due to endogenous and exogenous factors may affect the normal function of DNA-PK, which in turn could impair DNA repair and contribute to genomic instability. Recent studies implicate a role for epigenetics in the regulation of DNA-PK expression in normal and cancer cells, which may impact cancer progression and metastasis as well as provide opportunities for treatment and use of DNA-PK as a novel cancer biomarker. In addition, several small molecules and biological agents have been recently identified that can inhibit DNA-PK function or expression, and thus hold promise for cancer treatments. This review discusses the impact of epigenetic alterations and the expression of DNA-PK in relation to the DNA repair mechanisms with a focus on its differential levels in normal and cancer cells. [ABSTRACT FROM AUTHOR]

Though telomeres play a crucial role in maintaining genomic stability in cancer cells and have emerged as attractive therapeutic targets in anticancer therapy, the relationship between telomere dysfunction and genomic instability induced by irradiation is still unclear. In this study, we identified that protection of telomeres 1 (POT1), a single-stranded DNA (ssDNA)-binding protein, was upregulated in γ-irradiated HeLa cells and in cancer patients who exhibit radiation tolerance. Knockdown of POT1 delayed the repair of radiation-induced telomeric DNA damage which was associated with enhanced H3K9 trimethylation and enhanced the radiosensitivity of HeLa cells. The depletion of POT1 also resulted in significant genomic instability, by showing a significant increase in end-to-end chromosomal fusions, and the formation of anaphase bridges and micronuclei. Furthermore, knockdown of POT1 disturbed telomerase recruitment to telomere, and POT1 could interact with phosphorylated ATM (p-ATM) and POT1 depletion decreased the levels of p-ATM induced by irradiation, suggesting that POT1 could regulate the telomerase recruitment to telomeres to repair irradiation-induced telomeric DNA damage of HeLa cells through interactions with p-ATM. The enhancement of radiosensitivity in cancer cells can be achieved through the combination of POT1 and telomerase inhibitors, presenting a potential approach for radiotherapy in cancer treatment. • POT1 mitigates radiation-induced telomere damage and impacts histone condensation. • POT1 plays a key role in p-ATM-mediated recruitment of telomerase to telomeres. • Co-inhibition of telomerase and POT1 notably enhances tumor radiosensitivity. [ABSTRACT FROM AUTHOR]

Highlights • In ΔrecB cells DNA degradation is triggered at forks stalled at rrn operons. • No such degradation is observed in cells lacking Rep helicase. • In ΔrecB cells DNA degradation is triggered in the termination area. • SbcCD is the nuclease mostly responsible for degradation in both scenarios. • In ΔrecB cells degradation can occur at both ends of a linear chromosome. Abstract Bacterial chromosome duplication is initiated at a single origin (oriC). Two forks are assembled and proceed in opposite directions with high speed and processivity until they fuse and terminate in a specialised area opposite to oriC. Proceeding forks are often blocked by tightly-bound protein-DNA complexes, topological strain or various DNA lesions. In Escherichia coli the RecBCD protein complex is a key player in the processing of double-stranded DNA (dsDNA) ends. It has important roles in the repair of dsDNA breaks and the restart of forks stalled at sites of replication-transcription conflicts. In addition, ΔrecB cells show substantial amounts of DNA degradation in the termination area. In this study we show that head-on encounters of replication and transcription at a highly-transcribed rrn operon expose fork structures to degradation by nucleases such as SbcCD. SbcCD is also mostly responsible for the degradation in the termination area of ΔrecB cells. However, additional processes exacerbate degradation specifically in this location. Replication profiles from ΔrecB cells in which the chromosome is linearized at two different locations highlight that the location of replication termination can have some impact on the degradation observed. Our data improve our understanding of the role of RecBCD at sites of replication-transcription conflicts as well as the final stages of chromosome duplication. However, they also highlight that current models are insufficient and cannot explain all the molecular details in cells lacking RecBCD. [ABSTRACT FROM AUTHOR]

Cell cycle and apoptosis regulator 2 (CCAR2 or DBC1) is a human protein recently emerged as a novel and important player of the DNA damage response (DDR). Indeed, upon genotoxic stress, CCAR2, phosphorylated by the apical DDR kinases ATM and ATR, increases its binding to the NAD+-dependent histone deacetylase SIRT1 and inhibits SIRT1 activity. This event promotes the acetylation and activation of p53, a SIRT1 target, and the subsequent induction of p53 dependent apoptosis. In addition, CCAR2 influences DNA repair pathway choice and promotes the chromatin relaxation necessary for the repair of heterochromatic DNA lesions. However, besides DDR, CCAR2 is involved in several other cellular functions. Indeed, through the interaction with transcription factors, nuclear receptors, epigenetic modifiers and RNA polymerase II, CCAR2 regulates transcription and transcript elongation. Moreover, promoting Rev-erbα protein stability and repressing BMAL1 and CLOCK expression, it was reported to modulate the circadian rhythm. Through SIRT1 inhibition, CCAR2 is also involved in metabolism control and, suppressing RelB and p65 activities in the NFkB pathway, it restricts B cell proliferation and immunoglobulin production. Notably, CCAR2 expression is deregulated in several tumors and, compared to the non-neoplastic counterpart, it may be up- or down-regulated. Since its up-regulation in cancer patients is usually associated with poor prognosis and its depletion reduces cancer cell growth in vitro, CCAR2 was suggested to act as a tumor promoter. However, there is also evidence that CCAR2 functions as a tumor suppressor and therefore its role in cancer formation and progression is still unclear. In this review we discuss CCAR2 functions in the DDR and its multiple biological activities in unstressed cells. [ABSTRACT FROM AUTHOR]

Exposure of cells to genotoxic agents causes modifications in DNA, resulting to alterations in the genome. To reduce genomic instability, cells have DNA damage responses in which DNA repair proteins remove these lesions. Excessive free radicals cause DNA damages, repaired by base excision repair and nucleotide excision repair pathways. When non-oxidative lesions occur, genomic stability is maintained through checkpoints in which the cell cycle stops and DNA repair occurs. Telomere shortening is related to the development of various diseases, such as cancer. Low power lasers are used for treatment of a number of diseases, but they are also suggested to cause DNA damages at sub-lethal levels and alter transcript levels from DNA repair genes. This review focuses on genomic and telomere stabilization modulation as possible targets to improve therapeutic protocols based on low power lasers. Several studies have been carried out to evaluate the laser-induced effects on genome and telomere stabilization suggesting that exposure to these lasers modulates DNA repair mechanisms, telomere maintenance and genomic stabilization. Although the mechanisms are not well understood yet, low power lasers could be effective against DNA harmful agents by induction of DNA repair mechanisms and modulation of telomere maintenance and genomic stability. [ABSTRACT FROM AUTHOR]

DNA mismatch repair (MMR) is an important DNA repair pathway that plays critical roles in DNA replication fidelity, mutation avoidance and genome stability, all of which contribute significantly to the viability of cells and organisms. MMR is widely-used as a diagnostic biomarker for human cancers in the clinic, and as a biomarker of cancer susceptibility in animal model systems. Prokaryotic MMR is well-characterized at the molecular and mechanistic level; however, MMR is considerably more complex in eukaryotic cells than in prokaryotic cells, and in recent years, it has become evident that MMR plays novel roles in eukaryotic cells, several of which are not yet well-defined or understood. Many MMR-deficient human cancer cells lack mutations in known human MMR genes, which strongly suggests that essential eukaryotic MMR components/cofactors remain unidentified and uncharacterized. Furthermore, the mechanism by which the eukaryotic MMR machinery discriminates between the parental (template) and the daughter (nascent) DNA strand is incompletely understood and how cells choose between the EXO1-dependent and the EXO1–independent subpathways of MMR is not known. This review summarizes recent literature on eukaryotic MMR, with emphasis on the diverse cellular roles of eukaryotic MMR proteins, the mechanism of strand discrimination and cross-talk/interactions between and co-regulation of MMR and other DNA repair pathways in eukaryotic cells. The main conclusion of the review is that MMR proteins contribute to genome stability through their ability to recognize and promote an appropriate cellular response to aberrant DNA structures, especially when they arise during DNA replication. Although the molecular mechanism of MMR in the eukaryotic cell is still not completely understood, increased used of single-molecule analyses in the future may yield new insight into these unsolved questions. [ABSTRACT FROM AUTHOR]

NAD + is an essential cofactor for enzymes catalyzing redox-reactions as well as an electron carrier in energy metabolism. Aside from this, NAD + consuming enzymes like poly(ADP-ribose) polymerases and sirtuins are important regulators involved in chromatin-restructuring processes during repair and epigenetics/transcriptional adaption. In order to replenish cellular NAD + levels after cleavage, synthesis starts from precursors such as nicotinamide, nicotinamide riboside or nicotinic acid to match the need for this essential molecule. In the present study, we investigated the impact of supplementation with nicotinic acid on resting and proliferating human mononuclear blood cells with a focus on DNA damage and repair processes. We observed that nicotinic acid supplementation increased NAD + levels as well as DNA repair efficiency and enhanced genomic stability evaluated by micronucleus test after x-ray treatment. Interestingly, resting cells displayed lower basal levels of DNA breaks compared to proliferating cells, but break-induction rates were identical. Despite similar levels of p53 protein upregulation after irradiation, higher NAD + concentrations led to reduced acetylation of this protein, suggesting enhanced SIRT1 activity. Our data reveal that even in normal primary human cells cellular NAD + levels may be limiting under conditions of genotoxic stress and that boosting the NAD + system with nicotinic acid can improve genomic stability. [ABSTRACT FROM AUTHOR]

Cellular response to DNA damage, including ionizing radiation (IR) and UV radiation, is critical for the maintenance of genomic fidelity. Defects of DNA repair often result in genomic instability and malignant cell transformation. Centrosomal protein Nlp (ninein-like protein) has been characterized as an important cell cycle regulator that is required for proper mitotic progression. In this study, we demonstrate that Nlp is able to improve nucleotide excision repair (NER) activity and protects cells against UV radiation. Upon exposure of cells to UVC, Nlp is translocated into the nucleus. The C-terminus (1030-1382) of Nlp is necessary and sufficient for its nuclear import. Upon UVC radiation, Nlp interacts with XPA and ERCC1, and enhances their association. Interestingly, down-regulated expression of Nlp is found to be associated with human skin cancers, indicating that dysregulated Nlp might be related to the development of human skin cancers. Taken together, this study identifies mitotic protein Nlp as a new and important member of NER pathway and thus provides novel insights into understanding of regulatory machinery involved in NER. [ABSTRACT FROM AUTHOR]

The article discusses a study which investigated the chromosomal stability of human mesenchymal stromal cells (hMSCs) during cell culture and therapy.

Genomic instability and altered metabolism are key features of most cancers. Recent studies suggest that metabolic reprogramming is part of a systematic response to cellular DNA damage. Thus, defining the molecules that fine-tune metabolism in response to DNA damage will enhance our understanding of molecular mechanisms of tumorigenesis and have profound implications for the development of strategies for cancer therapy. Sirtuins have been established as critical regulators in cellular homeostasis and physiology. Here, we review the emerging data revealing a pivotal function of sirtuins in genome maintenance and cell metabolism, and highlight current advances about the phenotypic consequences of defects in these critical regulators in tumorigenesis. While many questions should be addressed about the regulation and context-dependent functions of sirtuins, it appears clear that sirtuins may provide a promising, exciting new avenue for cancer therapy. [ABSTRACT FROM AUTHOR]

The RECQ4 protein belongs to the RecQ helicase family, which plays crucial roles in genome maintenance. Mutations in the RECQ4 gene are associated with three insidious hereditary disorders: Rothmund–Thomson, Baller–Gerold, and RAPADILINO syndromes. These syndromes are characterized by growth deficiency, radial ray defects, red rashes, and higher predisposition to malignancy, especially osteosarcomas. Within the RecQ family, RECQ4 is the least characterized, and its role in DNA replication and repair remains unknown. We have identified several DNA binding sites within RECQ4. Two are located at the N-terminus and one is located within the conserved helicase domain. N-terminal domains probably cooperate with one another and promote the strong annealing activity of RECQ4. Surprisingly, the region spanning 322–400 aa shows a very high affinity for branched DNA substrates, especially Holliday junctions. This study demonstrates biochemical activities of RECQ4 that could be involved in genome maintenance and suggest its possible role in processing replication and recombination intermediates. [ABSTRACT FROM AUTHOR]

Longevity as a complex life-history trait shares an ontogenetic relationship with other quantitative traits and varies among individuals, families and populations. Heritability estimates of longevity suggest that about a third of the phenotypic variation associated with the trait is attributable to genetic factors, and the rest is influenced by epigenetic and environmental factors. Individuals react differently to the environments that they are a part of, as well as to the environments they construct for their survival and reproduction; the latter phenomenon is known as niche construction. Lifestyle influences longevity at all the stages of development and levels of human diversity. Hence, lifestyle may be viewed as a component of niche construction. Here, we: a) interpret longevity using a combination of genotype-epigenetic-phenotype (GEP) map approach and niche-construction theory, and b) discuss the plausible influence of genetic and epigenetic factors in the distribution and maintenance of longevity among individuals with normal life span on the one hand, and centenarians on the other. Although similar genetic and environmental factors appear to be common to both of these groups, exceptional longevity may be influenced by polymorphisms in specific genes, coupled with superior genomic stability and homeostatic mechanisms, maintained by negative frequency-dependent selection. We suggest that a comparative analysis of longevity between individuals with normal life span and centenarians, along with insights from population ecology and evolutionary biology, would not only advance our knowledge of biological mechanisms underlying human longevity, but also provide deeper insights into extending healthy life span. [ABSTRACT FROM AUTHOR]

The generation of induced pluripotent stem cells (iPSCs) often causes genetic and epigenetic defects, which may limit their clinical applications. Here, we show that reprogramming in the presence of small molecules preserved the genomic stability of iPSCs by inhibiting DNA double-strand breaks (DSBs) and activating Zscan4 gene. Surprisingly, the small molecules protected normal karyotype by facilitating repair of the DSBs that occurred during the early reprogramming process and long-term culture of iPSCs. The stemness and cell growth of iPSCs(+) were normally sustained with high expression of pluripotency genes compared that of iPSCs(−). Moreover, small molecules maintained the differentiation potential of iPSCs(+) for the three germ layers, whereas it was lost in iPSCs(−). Our results demonstrate that the defined small molecules are potent factors for generation of high quality iPSCs with preservation of genomic integrity by facilitating the reprogramming process. [ABSTRACT FROM AUTHOR]

Slow growth and genomic instability are the main problems facing tetraploid oyster breeding. In response to the above problem, we developed tetraploid by using Crassostrea gigas 'Haida No.3', a new variety with rapid growth and black shell color, and analyzed the growth, survival and ploidy of diploid ♀ × diploid ♂ (DD), diploid ♀ × tetraploid ♂ (DT: triploid) and tetraploid ♀ × tetraploid ♂(TT) in different periods. The results showed that there was no significant difference between the fertilization rate of DD, DT and TT, but the hatching rate of TT was significantly lower than that of DD (P < 0.05). The shell height of TT was significantly lower than DD, and the shell height of DT was significantly higher than that of DD at 13 days (P < 0.05). The survival rate of TT was lower than that of DD and DT in larval stage (P < 0.05), while there was no significant difference in the mean survival rate of DD, DT and TT in juvenile stage. The tetraploid rate in TT was 100% during the larval and juvenile stages. The shell color of all progeny in TT and DT was black and consistent with C. gigas 'Haida No.3'. These results indicated that black shell color of C. gigas 'Haida No.3' could be stably passed on to triploid and tetraploid offspring, suggesting that establishing genomically stable tetraploid populations was feasible by screening tetraploid parents. • The growth, survival and ploidy of diploid, triploid and tetraploid Pacific oysters were examined. • The growth rate of triploid (diploid ♀ ´ tetraploid ♂) was higher than that of diploid and tetraploid from day 13. • Black-shell color trait could be inherited stably in tetraploid offspring. [ABSTRACT FROM AUTHOR]

Highlights: [•] The spindle checkpoint detects unattached or tensionless kinetochores during mitosis and delays anaphase onset to ensure the fidelity of chromosome segregation. [•] Recent progress on the molecular understanding of the spindle checkpoint is reviewed. [•] The prominent roles of regulated protein–protein interactions and ubiquitin-dependent proteolysis in checkpoint signaling are highlighted. [•] The contributions of structural biology to the current understanding and future dissection of the spindle checkpoint are emphasized. [Copyright &y& Elsevier]

Abstract: Helicases have important roles in nucleic acid metabolism, and their prominence is marked by the discovery of genetic disorders arising from disease-causing mutations. Missense mutations can yield unique insight to molecular functions and basis for disease pathology. XPB or XPD missense mutations lead to Xeroderma pigmentosum, Cockayne''s syndrome, Trichothiodystrophy, or COFS syndrome, suggesting that DNA repair and transcription defects are responsible for clinical heterogeneity. Complex phenotypes are also observed for RECQL4 helicase mutations responsible for Rothmund–Thomson syndrome, Baller–Gerold syndrome, or RAPADILINO. Bloom''s syndrome causing missense mutations are found in the conserved helicase and RecQ C-terminal domain of BLM that interfere with helicase function. Although rare, patient-derived missense mutations in the exonuclease or helicase domain of Werner syndrome protein exist. Characterization of WRN separation-of-function mutants may provide insight to catalytic requirements for suppression of phenotypes associated with the premature aging disorder. Characterized FANCJ missense mutations associated with breast cancer or Fanconi anemia interfere with FANCJ helicase activity required for DNA repair and the replication stress response. For example, a FA patient-derived mutation in the FANCJ Iron-Sulfur domain was shown to uncouple its ATPase and translocase activity from DNA unwinding. Mutations in DDX11 (ChlR1) are responsible for Warsaw Breakage syndrome, a recently discovered autosomal recessive cohesinopathy. Ongoing and future studies will address clinically relevant helicase mutations and polymorphisms, including those that interfere with key protein interactions or exert dominant negative phenotypes (e.g., certain mutant alleles of Twinkle mitochondrial DNA helicase). Chemical rescue may be an approach to restore helicase activity in loss-of-function helicase disorders. Genetic and biochemical analyses of disease-causing missense mutations in human helicase disorders have led to new insights to the molecular defects underlying aberrant cellular and clinical phenotypes. [Copyright &y& Elsevier]

Abstract: Selenium (Se) is an essential micronutrient for humans, acting as a component of the unusual amino acids, selenocysteine (Se-Cys) and selenomethionine (Se-Met). Where Se levels are low, the cell cannot synthesise selenoproteins, although some selenoproteins and some tissues are prioritised over others. Characterised functions of known selenoproteins, include selenium transport (selenoprotein P), antioxidant/redox properties (glutathione peroxidases (GPxs), thioredoxin reductases and selenoprotein P) and anti-inflammatory properties (selenoprotein S and GPx4). Various forms of Se are consumed as part of a normal diet, or as a dietary supplement. Supplementation of tissue culture media, animal or human diets with moderate levels of certain Se compounds may protect against the formation of DNA adducts, DNA or chromosome breakage, and chromosome gain or loss. Protective effects have also been shown on mitochondrial DNA, and on telomere length and function. Some of the effects of Se compounds on gene expression may relate to modulation of DNA methylation or inhibition of histone deacetylation. Despite a large number of positive effects of selenium and selenoproteins in various model systems, there have now been some human clinical trials that have shown adverse effects of Se supplementation, according to various endpoints. Too much Se is as harmful as too little, with animal models showing a “U”-shaped efficacy curve. Current recommended daily allowances differ among countries, but are generally based on the amount of Se necessary to saturate GPx enzymes. However, increasing evidence suggests that other enzymes may be more important than GPx for Se action, that optimal levels may depend upon the form of Se being ingested, and vary according to genotype. New paradigms, possibly involving nutrigenomic tools, will be necessary to optimise the forms and levels of Se desirable for maximum protection of genomic stability in all humans. [Copyright &y& Elsevier]

Abstract: Zinc (Zn) is an essential trace element required for maintaining both optimal human health and genomic stability. Zn plays a critical role in the regulation of DNA repair mechanisms, cell proliferation, differentiation and apoptosis involving the action of various transcriptional factors and DNA or RNA polymerases. Zn is an essential cofactor or structural component for important antioxidant defence proteins and DNA repair enzymes such as Cu/Zn SOD, OGG1, APE and PARP and may also affect activities of enzymes such as BHMT and MTR involved in methylation reactions in the folate-methionine cycle. This review focuses on the role of Zn in the maintenance of genome integrity and the effects of deficiency or excess on genomic stability events and cell death. [Copyright &y& Elsevier]

Abstract: Coenzyme Q10 (CoQ10) is an essential component for electron transport in the mitochondrial respiratory chain and serves as cofactor in several biological processes. The reduced form of CoQ10 (ubiquinol, Q10H2) is an effective antioxidant in biological membranes. During the last years, particular interest has been grown on molecular effects of CoQ10 supplementation on mechanisms related to DNA damage prevention. This review describes recent advances in our understanding about the impact of CoQ10 on genomic stability in cells, animals and humans. With regard to several in vitro and in vivo studies, CoQ10 provides protective effects on several markers of oxidative DNA damage and genomic stability. In comparison to the number of studies reporting preventive effects of CoQ10 on oxidative stress biomarkers, CoQ10 intervention studies in humans with a direct focus on markers of DNA damage are limited. Thus, more well-designed studies in healthy and disease populations with long-term follow up results are needed to substantiate the reported beneficial effects of CoQ10 on prevention of DNA damage. [Copyright &y& Elsevier]

Abstract: Model systems have been extremely useful for studying various theories of aging. Studies of yeast have been particularly helpful to explore the molecular mechanisms and pathways that affect aging at the cellular level in the simple eukaryote. Although genetic analysis has been useful to interrogate the aging process, there has been both interest and debate over how functionally conserved the mechanisms of aging are between yeast and higher eukaryotes, especially mammalian cells. One area of interest has been the importance of genomic stability for age-related processes, and the potential conservation of proteins and pathways between yeast and human. Translational genetics have been employed to examine the functional roles of mammalian proteins using yeast as a pliable model system. In the current review recent advancements made in this area are discussed, highlighting work which shows that the cellular functions of human proteins in DNA repair and maintenance of genomic stability can be elucidated by genetic rescue experiments performed in yeast. [Copyright &y& Elsevier]

Abstract: Background: Peri-conceptional use of folic acid contributes to protection against congenital malformations, such as neural tube defects and cleft lip with or without cleft palate (CL/P). Previous studies showed that low folate levels cause DNA damage, leading to chromosomal instability and aneusomy. This study seeks to confirm this finding and investigates whether the in vitro sensitivity towards aneusomy of chromosome 17 and 21 in the folate-deficient state differs between CL/P patients and controls. Methods: Epstein–Barr virus-immortalized B-lymphoblasts derived from 15 CL/P children and 15 controls, were cultured in medium with high and low concentrations – approximately 40nM and 5nM – of 5-methyltetrahydrofolate, respectively. Fluorescence in situ hybridization was used to detect specific fluorescence signals for chromosomes 17 and 21. Results: A significant increase in aneusomy of chromosomes 17 (2.3% vs 7.6%; p ≤0.001) and 21 (2.5% vs 7.0%; p ≤0.001) was observed after 10 days of culturing in low folate. These results were comparable in cell lines from patients and controls. Interestingly, for chromosome 17 the folate deficiency mainly resulted in an increase of monosomy (6%, p ≤0.001), while for chromosome 21 the increase of trisomy was larger (4.9%, p ≤0.001). Conclusions: These data suggest that folate deficiency is a significant risk factor in the development of aneusomy and may affect the distribution of chromosomes during cell division. The comparable aneusomy frequencies in CL/P and in controls suggest that other folate-related processes are involved in the pathogenesis of CL/P, and additional investigations are needed to identify the causal mechanisms. [Copyright &y& Elsevier]

Abstract: The tumor suppressor p53 is a transcription factor whose function is critical for maintaining genomic stability in mammalian cells. In response to DNA damage, p53 initiates a signaling cascade that results in cell cycle arrest, DNA repair or, if the damage is severe, programmed cell death. In addition, p53 interacts with repair proteins involved in homologous recombination. Mitotic homologous recombination (HR) plays an essential role in the repair of double-strand breaks (DSBs) and broken replication forks. Loss of function of either p53 or HR leads to an increased risk of cancer. Given the importance of both p53 and HR in maintaining genomic integrity, we analyzed the effect of p53 on HR in vivo using Fluorescent Yellow Direct Repeat (FYDR) mice as well as with the sister chromatid exchange (SCE) assay. FYDR mice carry a direct repeat substrate in which an HR event can yield a fluorescent phenotype. Here, we show that p53 status does not significantly affect spontaneous HR in adult pancreatic cells in vivo or in primary fibroblasts in vitro when assessed using the FYDR substrate and SCEs. In addition, primary fibroblasts from p53 null mice do not show increased susceptibility to DNA damage-induced HR when challenged with mitomycin C. Taken together, the FYDR assay and SCE analysis indicate that, for some tissues and cell types, p53 status does not greatly impact HR. [Copyright &y& Elsevier]

Abstract: Cancer cells utilize complex mechanisms to remodel their bioenergetic properties. We exploited the intrinsic genomic stability of xeroderma pigmentosum C (XPC) to understand the inter-relationships between genomic instability, reactive oxygen species (ROS) generation, and metabolic alterations during neoplastic transformation. We showed that knockdown of XPC (XPCKD) in normal human keratinocytes results in metabolism remodeling through NADPH oxidase-1 (NOX-1) activation, which in turn leads to increased ROS levels. While enforcing antioxidant defenses by overexpressing catalase, CuZnSOD, or MnSOD could not block the metabolism remodeling, impaired NOX-1 activation abrogates both alteration in ROS levels and modifications of energy metabolism. As NOX-1 activation is observed in human squamous cell carcinomas (SCCs), the blockade of NOX-1 could be a target for the prevention and the treatment of skin cancers. This article is part of a Special Issue entitled: Bioenergetics of Cancer. [Copyright &y& Elsevier]

Abstract: Fanconi anemia (FA) is an inherited disease characterized by bone marrow failure, increased cancer risk and hypersensitivity to DNA cross-linking agents, implying a role for this pathway in the maintenance of genomic stability. The central player of the FA pathway is the multi-subunit E3 ubiquitin ligase complex activated through a replication- and DNA damage-dependent mechanism. A consequence of the activation of the complex is the monoubiquitylation of FANCD2 and FANCI, late term effectors in the maintenance of genome integrity. The details regarding the coordination of the FA-dependent response and the DNA replication process are still mostly unknown. We found, by yeast two-hybrid assay and co-immunoprecipitation in human cells, that the core complex subunit FANCF physically interacts with PSF2, a member of the GINS complex essential for both the initiation and elongation steps of DNA replication. In HeLa cells depleted for PSF2, we observed a decreased binding to chromatin of the FA core complex, suggesting that the GINS complex may have a role in either loading or stabilizing the FA core complex onto chromatin. Consistently, GINS and core complex bind chromatin contemporarily upon origin firing and PSF2 depletion sensitizes cells to DNA cross-linking agents. However, depletion of PSF2 is not sufficient to reduce monoubiquitylation of FANCD2 or its localization to nuclear foci following DNA damage. Our results suggest a novel crosstalk between DNA replication and the FA pathway. [Copyright &y& Elsevier]

Abstract: Apurinic/apyrimidinic (AP) endonucleases are versatile DNA repair enzymes that possess a variety of nucleolytic activities, including endonuclease activity at AP sites, 3′ phosphodiesterase activity that can remove a variety of ligation-blocking lesions from the 3′ end of DNA, endonuclease activity on oxidative DNA lesions, and 3′ to 5′ exonuclease activity. There are two families of AP endonucleases, named for the bacterial counterparts endonuclease IV (EndoIV) and exonuclease III (ExoIII). While ExoIII family members are present in all kingdoms of life, EndoIV members exist in lower organisms but are curiously absent in plants, mammals and some other vertebrates. Here, we review recent research on these enzymes, focusing primarily on the EndoIV family. We address the role(s) of EndoIV members in DNA repair and discuss recent findings from each model organism in which the enzymes have been studied to date. [ABSTRACT FROM AUTHOR]

Abstract: The X family of DNA polymerases in eukaryotic cells consists of terminal transferase and DNA polymerases β, λ, and μ. These enzymes have similar structural portraits, yet different biochemical properties, especially in their interactions with DNA. None of these enzymes possesses a proofreading subdomain, and their intrinsic fidelity of DNA synthesis is much lower than that of a polymerase that functions in cellular DNA replication. In this review, we discuss the similarities and differences of three members of Family X: polymerases β, λ, and μ. We focus on biochemical mechanisms, structural variation, fidelity and lesion bypass mechanisms, and cellular roles. Remarkably, although these enzymes have similar three-dimensional structures, their biochemical properties and cellular functions differ in important ways that impact cellular function. [Copyright &y& Elsevier]

Abstract: The class III histone deacetylase (HDAC) SIRT1 plays a role in the metabolism, aging, and carcinogenesis of organisms and regulates senescence and apoptosis in cells. Recent reports revealed that SIRT1 also deacetylates several DNA double-strand break (DSB) repair proteins. However, its exact functions in DNA repair remained elusive. Using nuclear foci analysis and fluorescence-based, chromosomal DSB repair reporter, we find that SIRT1 activity promotes homologous recombination (HR) in human cells. Importantly, this effect is unrelated to functions of poly(ADP-ribose) polymerase 1 (PARP1), another NAD(+)-catabolic protein, and does not correlate with cell cycle changes or apoptosis. Interestingly, we demonstrate that inactivation of Rad51 does not eliminate the effect of SIRT1 on HR. By epistasis-like analysis through knockdown and use of mutant cells of distinct SIRT1 target proteins, we show that the non-homologous end joining (NHEJ) factor Ku70 as well as the Nijmegen Breakage Syndrome protein (nibrin) are not needed for this SIRT1-mediated effect, even though a partial contribution of nibrin cannot be excluded. Strikingly however, the Werner helicase (WRN), which in its mutated form causes premature aging and cancer and which was linked to the Rad51-independent single-strand annealing (SSA) DSB repair pathway, is required for SIRT1-mediated HR. These results provide first evidence that links SIRT1''s functions to HR with possible implications for genomic stability during aging and tumorigenesis. [Copyright &y& Elsevier]

Abstract: Stem cells have been described in most adult tissues, where they play a key role in maintaining tissue homeostasis. As they self-renew throughout life, accumulating genetic anomalies can compromise their genomic integrity and potentially give rise to cancer. Stem cells (SCs) may thus be a major target of radiation carcinogenesis. In addition, unrepaired genotoxic damage may cause cell death and stem cell pool depletion, impairing lineage functionality and accelerating aging. Developments in SC biology enabled the characterization of the responses of stem cells to genotoxic stress and their role in tissue damage. We here examine how these cells react to ionizing radiation (IR), and more specifically their radiosensitivity, stress signaling and DNA repair. We first review embryonic SCs, as a paradigm of primitive pluripotent cells, then three adult tissues, bone marrow, skin and intestine, capable of long-term regeneration and at high risk for acute radiation syndromes and long-term carcinogenesis. We discuss IR disruption of the fine balance between maintenance of tissue homeostasis and genomic stability. We show that stem cell radiosensitivity does not follow a unique model, but differs notably according to the turnover rates of the tissues. [Copyright &y& Elsevier]

Abstract: Five human RecQ helicases (WRN, BLM, RECQ4, RECQ5, RECQ1) exist in humans. Of these, three are genetically linked to diseases of premature aging and/or cancer. Neither RECQ1 nor RECQ5 has yet been implicated in a human disease. However, cellular studies and genetic analyses of model organisms indicate that RECQ1 (and RECQ5) play an important role in the maintenance of genomic stability. Biochemical studies of purified RECQ1 protein demonstrate that the enzyme catalyzes DNA unwinding and strand annealing, and these activities are likely to be important for its role in DNA repair. RECQ1 also physically and functionally interacts with proteins involved in genetic recombination. In this review, we will summarize our current knowledge of RECQ1 roles in cellular nucleic acid metabolism and propose avenues of investigation for future studies. [Copyright &y& Elsevier]

Long non-coding RNAs (lncRNAs) are a fascinating, but still largely uncharacterized, class of genes. A recent paper by the Mendell group identifies NORAD, a novel lncRNA that is regulated in response to DNA damage and plays a key role in maintaining genome integrity by modulating the activity the RNA binding proteins PUM2 and PUM1. [ABSTRACT FROM AUTHOR]

Abstract: Human ESCs provide an opportunity for modeling human-specific strategies to study the earliest events leading to normal hematopoietic specification versus leukemic transformation. Of interest, are the human childhood acute leukemias harboring specific fusion oncogenes such as MLL-AF4, TEL-AML1 or BCR-ABL wherein clinically significant manifestations arise in utero. The mechanisms of transformation are not amenable to analysis with patient samples and, many mouse models for pediatric leukemias have fallen short in illuminating the human disease because they do not recapitulate key aspects of the actual disease, suggesting that the mouse models are missing essential components of oncogenesis present in the human embryo. Prior to using hESCs as a tentative system for modeling leukemia, robust studies aimed at demonstrating their genetic stability are required; otherwise, cooperating mutations already present could prime hESCs susceptible to transformation. We performed an extensive molecular cytogenetic and cellular in vitro and in vivo analysis which reveals an overall genomic stability of HS181 and HS293 hESCs maintained long-term by mechanical dissociation in human feeders. Importantly, we show for the first time that the genetically stable HS181 hESC line differentiates into CD45+ hematopoietic cells and clonogenic hematopoietic progenitors. This data should encourage stem cell researchers to implement robust cytogenetic tools when assessing hESC genetic stability, in order to detect tiny but relevant biological functional or structural chromosome abnormalities and, paves the way for generating fusion oncogene-expressing transgenic hESCs as a human-specific system for studying the early in utero events leading to normal hematopoietic specification versus childhood leukemic transformation. [Copyright &y& Elsevier]

Abstract: TREX2 is a 3′→5′ exonuclease that binds to DNA and removes 3′ mismatched nucleotides. By an in vitro structure function analysis, we found a single amino acid change (H188A) completely ablates exonuclease activity and impairs DNA binding by about 60% while another change (R167A) impairs DNA binding by about 85% without impacting exonuclease activity. For a biological analysis, we generated trex2 null cells by deleting the entire Trex2 coding sequences in mouse embryonic stem (ES) cells. We found Trex2 deletion caused high levels of Robertsonian translocations (RbTs) showing Trex2 is important for chromosomal maintenance. Here we evaluate the exonuclease and DNA binding domains by expressing in trex2 null cells coding sequences for wild type human TREX2 (Trex2 hTX2 ) or human TREX2 with the H188A change (Trex2 H188A ) or the R167A change (Trex2 R167A ). These cDNAs are positioned adjacent to the mouse Trex2 promoter by Cre-mediated knock-in. By observing metaphase spreads, we found Trex2 H188A cells exhibited high levels of double-strand breaks (DSBs) and chromosomal fragments. Therefore, TREX2 may suppress spontaneous DSBs or exonuclease defective TREX2 may induce them in a dominate-negative manner. We also found Trex2 hTX2 , hTrex2 H188A and hTrex2 R167A cells did not exhibit RbTs. Thus, neither the exonuclease nor DNA binding domains suppress RbTs suggesting TREX2 possesses additional biochemical activities. [Copyright &y& Elsevier]

Abstract: Maintaining the integrity of spermatogenic stem cells is essential to transfer genetic information to a descendant. However, knowledge of maintenance of genetic stability in stem cells is still limited. RAD18 is critical for postreplication repair through mono- and multi-ubiquitination of proliferating cell nuclear antigen (PCNA) to maintain genomic stability. Mammalian RAD18 is highly expressed in the spermatocytes and the nuclei of a few spermatogonia in adult mice. To elucidate the physiological function of RAD18, we analyzed a phenotype of Rad18−/− mice. The mice were born and appeared to grow normally. Although the mice were fertile, fertility and testis weight decreased with age. Histological examination revealed normal spermatogenesis in almost all seminiferous tubules in Rad18−/− testes at 2 months old, and abnormal sperm could not be detected in the epididymis. However, 25% of the tubules lost almost all germ cells at 12 months. The seminiferous tubules frequently retained only late differentiated phase germ cells, suggesting that the exhaustion of spermatogonial stem cells leads to the loss of all germ cells in the seminiferous tubules. Wild-type germ cells were successfully transplanted into and colonized in the seminiferous tubules of aged Rad18−/− mice, indicating that Sertoli cells have a normal supportive function even in aged testes. We conclude that RAD18 is intrinsically required for the long-term maintenance of spermatogenesis. [Copyright &y& Elsevier]

[Display omitted] The regulation of the apoptotic pathway is one of the most studied mechanisms regarding cancer cell resistance. Many mutations have been linked to drug resistance. The DNA fragmentation factor 40 (DFF40) has been gaining interest regarding cancer cell response to chemotherapy and patient outcomes. Glioblastomas and uterine leiomyosarcomas have been shown to have a downregulation of DFF40 expression, conferring a poor patient prognosis. In concordance with these observations, in this study, we showed that DFF40 gene is also downregulated in breast, endocervical, ovarian, lung, pancreas and glioblastomas. DFF40 is the endonuclease responsible of DNA fragmentation during apoptosis. In this study, we sought to determine if a DFF40 deficiency in Jurkat T cells could impact the sensitivity to conventional chemotherapy drugs. CRISPR-cas9 generated DFF40 knockout (DFF40 KO) stable Jurkat cells and wild-type (DFF40 WT) cells were treated with different antimetabolites and topoisomerase II (TOP2) inhibitors, and cell viability was subsequently assessed. DFF40 deficient cells show chemoresistance to antimetabolites (e.g. methotrexate, 6-mercaptopurine and cytarabine) and surprisingly, they are more sensitive to TOP2 inhibitors (e.g. etoposide and teniposide). DFF40 deficient cells exposed to cytarabine present lower phosphatidylserine translocation levels to the outer cell membrane layer. Etoposide exposure in DFF40 deficient cells induces higher mortality levels and downregulation of Bcl-xL cells compared to DFF40 expressing T cells. The abolition of DFF40 expression in Jurkat cells significantly impairs histone H2AX phosphorylation following etoposide and cytarabine treatments. Our findings suggest that DFF40 is a novel key target in cancer cell resistance that potentially regulates genomic stability. [ABSTRACT FROM AUTHOR]

Abstract: DNA damage response mechanisms encompass pathways of DNA repair, cell cycle checkpoint arrest and apoptosis. Together, these mechanisms function to maintain genomic stability in the face of exogenous and endogenous DNA damage. ATM is activated in response to double strand breaks and initiates cell cycle checkpoint arrest. Recent studies in human fibroblasts have shown that ATM also regulates a mechanism of end-processing that is required for a component of double strand break repair. Human fibroblasts rarely undergo apoptosis after ionising radiation and, therefore, apoptosis is not considered in our review. The dual function of ATM raises the question as to how the two processes, DNA repair and checkpoint arrest, interplay to maintain genomic stability. In this review, we consider the impact of ATM''s repair and checkpoint functions to the maintenance of genomic stability following irradiation in G2. We discuss evidence that ATM''s repair function plays little role in the maintenance of genomic stability following exposure to ionising radiation. ATM''s checkpoint function has a bigger impact on genomic stability but strikingly the two damage response pathways co-operate in a more than additive manner. In contrast, ATM''s repair function is important for survival post irradiation. [Copyright &y& Elsevier]

Abstract: The prominent expression of tenascin-C in the stroma of most solid tumors, first observed in the mid 1980s, implicates tenascin-C in tumorigenesis. This is also supported by in vitro experiments that demonstrate the capacity of tenascin-C to stimulate tumor growth by various mechanisms including promotion of proliferation, escaping immuno-surveillance and positively influencing angiogenesis. However, tumorigenesis in tenascin-C knock-out mice is not significantly different from that observed in control animals. Perhaps this is not unexpected if one considers that tenascin-C may act as an oncogene. The potential role of tenascin-C in tumorigenesis through its oncogenic action on cellular signaling will be discussed in this review, including how tenascin-C mediated tumor cell detachment might affect genome stability. [Copyright &y& Elsevier]

Abstract: The importance of poly(ADP-ribose) metabolism in the maintenance of genomic integrity following genotoxic stress has long been firmly established. Poly(ADP-ribose) polymerase-1 (PARP-1) and its catabolic counterpart, poly(ADP-ribose) glycohydrolase (PARG) play major roles in the modulation of cell responses to genotoxic stress. Recent discoveries of a number of other enzymes with poly(ADP-ribose) polymerase activity have established poly(ADP-ribosyl)ation as a general biological mechanism in higher eukaryotic cells that not only promotes cellular recovery from genotoxic stress and eliminates severely damaged cells from the organism, but also ensures accurate transmission of genetic information during cell division. Additionally, emerging data suggest the involvement of poly(ADP-ribosyl)ation in the regulation of intracellular trafficking, memory formation and other cellular functions. In this brief review on PARP and PARG enzymes, emphasis is placed on PARP-1, the best understood member of the PARP family and on the relationship of poly(ADP-ribosyl)ation to cancer and other diseases of aging. [Copyright &y& Elsevier]

Abstract: Genomic instability is characteristic of malignant cells, and a strong correlation exists between abnormal karyotype and tumorigenicity. Increased expression of the homologous recombination and DNA repair protein Rad51 has been reported in immortalized cell lines and multiple primary tumor cell types which could alter recombination pathways to contribute to the chromosomal rearrangements found in these cells. In addition, Rad51 participates in a complex network of interactions that includes DNA damage sensors, tumor suppressors, and cell cycle and apoptotic regulators, and mutation of many of these proteins have also been associated with tumor initiation or progression. Insights into the connection between disregulated Rad51 and malignant phenotype indicate that Rad51 is a potential target for new anti-cancer regimens including those that use siRNA technology. [Copyright &y& Elsevier]

Abstract: DNA double strand breaks represent the most threatening lesion to the integrity of the genome in cells exposed to ionizing radiation and radiomimetic chemicals. Those breaks are recognized, signaled to cell cycle checkpoints and repaired by protein complexes. The product of the gene (ATM) mutated in the human genetic disorder ataxia–telangiectasia (A–T) plays a central role in the recognition and signaling of DNA damage. ATM is one of an ever growing number of proteins which when mutated compromise the stability of the genome and predispose to tumour development. Mechanisms for recognising double strand breaks in DNA, maintaining genome stability and minimizing risk of cancer are discussed. [Copyright &y& Elsevier]

Nondisjunction of chromosomes results in aneuploidy in mammalian cells causing genomic instability. The spindle checkpoint, one of the surveillance systems to maintain genomic stability, prevents missegregation of chromosomes until all the kinetochores are properly attached with bipolar spindles. When this condition is not met, MAD2, a component of the spindle checkpoint complex, associates with p55CDC/hCDC20 to inhibit ubiquitination of substrates by the anaphase-promoting complex (APC). In this study, we have focused on the biological role of the MAD2-binding domain in p55CDC/hCDC20 in the maintenance of genomic stability. Based on previous studies, we constructed a truncated p55CDC/hCDC20 mutant (F2) that harbors only the MAD2-binding domain. Interestingly, we found that in the yeast two-hybrid system, the interaction of F2 and MAD2 was stronger than that of intact p55CDC/hCDC20. We also found that in the presence of the microtubule-disrupting drug, nocodazole, U2OS cells expressing p55CDC/hCDC20 mutants bypassed the mitotic arrest and showed apoptotic morphologies, whereas cells harboring vector alone arrested at metaphase. In particular, the apoptotic phenomena were dramatically enhanced in the F2-expressing cells. These mitotic catastrophes also occurred in cells treated with other microtubule disrupting agents, such as taxol and vinblastine. In addition, the mutant cells exhibited chromosomal missegregation during mitosis, even in the absence of nocodazole. Taken together, these results suggest that agents blocking the spindle checkpoint response may induce tumor cells to become more sensitive to spindle poison drugs, providing a powerful tool to improve chemotherapy. [Copyright &y& Elsevier]

The postreplication repair pathway (PRR) is composed of error-free and error-prone sub-pathways that allow bypass of DNA damage-induced replication-blocking lesions. The error-free sub-pathway is also used for bypass of spontaneous DNA damage and functions in cooperation with recombination pathways. In diploid yeast cells, error-free PRR is needed to prevent genomic instability, which is manifest as loss of heterozygosity (LOH) events of increased chromosome loss and recombination. Homologous recombination acts synergistically with the error-free damage avoidance branch of PRR to prevent chromosome loss. The DNA damage checkpoint gene MEC1 acts synergistically with the PRR pathway in maintaining genomic stability. Integration of the PRR pathway with other cellular pathways for preventing genomic instability is discussed. In diploid strains, the most dramatic increase is in the abnormality of chromosome loss when a repair or damage detection pathway is defective. [Copyright &y& Elsevier]

Ataxia-telangiectasia (AT) is an autosomal recessive disorder characterized by genomic instability, chronic oxidative damage, and increased cancer incidence. Compared to normal cells, AT cells exhibit unusual sensitivity to exogenous oxidants, including t-butyl hydroperoxide (t-BOOH). Since ferritin releases labile iron under oxidative stress (which is chronic in AT) and labile iron mediates the toxic effects of t-butyl hydroperoxide, we hypothesized that chelation of intracellular labile iron would increase the genomic stability of AT cells, with and without exogenous oxidative stress. Here we report that desferrioxamine treatment increases the plating efficiency of AT, but not normal cells, in the colony forming-efficiency assay (a method often used to measure genomic stability). Additionally, desferrioxamine increases AT, but not normal cell resistance, to t-butyl hydroperoxide in this assay. Last, AT cells exhibit increased sensitivity to the toxic effects of FeCl2 in the colony forming-efficiency assay and fail to demonstrate a FeCl2-induced G2 checkpoint response when compared to normal cells. Our data indicates that: (1) chelation of labile iron increases genomic stability in AT cells, but not normal cells; and (2) AT cells exhibit deficits in their responses to iron toxicity. While preliminary, our findings suggest that AT might be, in part, a disorder of iron metabolism and treatment of individuals with AT with desferrioxamine might have clinical efficacy. [Copyright &y& Elsevier]

Actin cytoskeleton remodeling is the major motor of cytoskeleton dynamics driving tumor cell adhesion, migration and invasion. The typical RhoA, RhoB and RhoC GTPases are the main regulators of actin cytoskeleton dynamics. The C3 exoenzyme transferase from Clostridium botulinum is a toxin that causes the specific ADP-ribosylation of Rho-like proteins, leading to its inactivation. Here, we examine what effects the Rho GTPase inhibition and the consequent actin cytoskeleton instability would have on the emergence of DNA damage and on the recovery of genomic stability of malignant melanoma cells, as well as on their survival. Therefore, the MeWo cell line, here assumed as a melanoma cell line model for the expression of genes involved in the regulation of the actin cytoskeleton, was transiently transfected with the C3 toxin and subsequently exposed to UV-radiation. Phalloidin staining of the stress fibers revealed that actin cytoskeleton integrity was strongly disrupted by the C3 toxin in association with reduced melanoma cells survival, and further enhanced the deleterious effects of UV light. MeWo cells with actin cytoskeleton previously perturbed by the C3 toxin still showed higher levels and accumulation of UV-damaged DNA (strand breaks and cyclobutane pyrimidine dimers, CPDs). The interplay between reduced cell survival and impaired DNA repair upon actin cytoskeleton disruption can be explained by constitutive ERK1/2 activation and an inefficient phosphorylation of DDR proteins (γH2AX, CHK1 and p53) caused by C3 toxin treatment. Altogether, these results support the general idea that actin network help to protect the genome of human cells from damage caused by UV light through unknown molecular mechanisms that tie the cytoskeleton to processes of genomic stability maintenance. • Specific targeting of Rho GTPases by C3 toxin disrupts actin cytoskeleton. • Genotoxic stress promoted by UV light affects the integrity of actin cytoskeleton. • C3 toxin hypersensitizes melanoma cells to the UV-radiation effects reducing survival. • The actin cytoskeleton integrity is directly linked to genomic stability. • C3 toxin associated with UV stress reduces DDR signaling an increases MAPK signaling. [ABSTRACT FROM AUTHOR]

Various culture systems have been used to derive and maintain human pluripotent stem cells (hPSCs), but they are inefficient in sustaining cloning and suspension expansion of hPSCs. Through systematically modulating Wnt and Activin/Nodal signaling, we developed a defined medium (termed AIC), which enables efficient cloning and long-term expansion of hPSCs (AIC-hPSCs) through single-cell passage on feeders, matrix or in suspension (25-fold expansion in 4 days) and maintains genomic stability of hPSCs over extensive expansion. Moreover, the AIC medium supports efficient derivation of hPSCs from blastocysts or somatic cells under feeder-free conditions. Compared to conventional hPSCs, AIC-hPSCs have similar gene expression profiles but down-regulated differentiation genes and display higher metabolic activity. Additionally, the AIC medium shows a good compatibility for different hPSC lines under various culture conditions. Our study provides a robust culture system for derivation, cloning and suspension expansion of high-quality hPSCs that benefits GMP production and processing of therapeutic hPSC products. [ABSTRACT FROM AUTHOR]