Structural Perspectives on Metal Dependent Roles of Ferric Uptake Regulator (Fur).

Iron is crucial for the metabolism and growth of most prokaryotic cells. The ferric uptake regulator (Fur) protein plays a central role in regulating iron homeostasis and metabolic processes in bacteria. It ensures the proper utilization of iron and the maintenance of cellular functions in response to environmental cues. Fur proteins are composed of an N-terminal DNA-binding domain (DBD) and a C-terminal dimerization domain (DD), typically existing as dimers in solution. Fur proteins have conserved metal-binding sites named S1, S2, and S3. Among them, site S2 serves as a regulatory site, and metal binding at S2 results in conformational changes. Additionally, as a transcriptional regulator, Fur specifically binds to a consensus DNA sequence called the Fur box. To elucidate the structural and functional properties of Fur proteins, various structures of metal- or DNA-bound Fur proteins or apo-Fur proteins have been determined. In this review, we focus on the structural properties of Fur proteins according to their ligand-bound state and the drug development strategies targeting Fur proteins. This information provides valuable insights for drug discovery.

Signification and Application of Mutator and Antimutator Phenotype-Induced Genetic Variations in Evolutionary Adaptation and Cancer Therapeutics.

Mutations present a dichotomy in their implications for cellular processes. They primarily arise from DNA replication errors or damage repair processes induced by environmental challenges. Cumulative mutations underlie genetic variations and drive evolution, yet also contribute to degenerative diseases such as cancer and aging. The mutator phenotype elucidates the heightened mutation rates observed in malignant tumors. Evolutionary adaptation, analogous to bacterial and eukaryotic systems, manifests through mutator phenotypes during changing environmental conditions, highlighting the delicate balance between advantageous mutations and their potentially detrimental consequences. Leveraging the genetic tractability of Saccharomyces cerevisiae offers unique insights into mutator phenotypes and genome instability akin to human cancers. Innovative reporter assays in yeast model organisms enable the detection of diverse genome alterations, aiding a comprehensive analysis of mutator phenotypes. Despite significant advancements, our understanding of the intricate mechanisms governing spontaneous mutation rates and preserving genetic integrity remains incomplete. This review outlines various cellular pathways affecting mutation rates and explores the role of mutator genes and mutation-derived phenotypes, particularly prevalent in malignant tumor cells. An in-depth comprehension of mutator and antimutator activities in yeast and higher eukaryotes holds promise for effective cancer control strategies.

Yap1-mediated Flr1 expression reveals crosstalk between oxidative stress signaling and caffeine resistance in Saccharomyces cerevisiae.

Caffeine, a methylxanthine derivative, affects various physiological conditions such as cell growth, proliferation, and energy metabolism. A genome-wide screening for genes required for caffeine resistance in Schizosaccharomyces pombe revealed several candidates, including Pap1 and downstream target genes involved in caffeine efflux. We found that Yap1, a budding yeast AP-1 homolog required for oxidative stress response, has a caffeine tolerance function. Although the Yap1 mutant is not sensitive to caffeine, overexpression of Yap1 renders cells resistant to high concentrations of caffeine. Caffeine sensitivity of mutants lacking two multidrug transporters, Pdr5 or Snq2, is completely recovered by Yap1 overexpression. Among Yap1-dependent target genes, FLR1, a fluconazole-resistant gene, is necessary but not sufficient for caffeine tolerance. Low concentrations of hydrogen peroxide induce Yap1 activation, which restores cell viability against caffeine toxicity. Intriguingly, oxidative stress-mediated cellular adaptation to caffeine toxicity requires Yap1, but not Flr1. Moreover, caffeine is involved in reduction of intracellular reactive oxygen species (ROS), as well as mutation rate and Rad52 foci formation. Altogether, we identified novel reciprocal crosstalk between ROS signaling and caffeine resistance.

Pleiotropic Effects of Caffeine Leading to Chromosome Instability and Cytotoxicity in Eukaryotic Microorganisms.

Caffeine, a methylxanthine analog of purine bases, is a compound that is largely consumed in beverages and medications for psychoactive and diuretic effects and plays many beneficial roles in neuronal stimulation and enhancement of anti-tumor immune responses by blocking adenosine receptors in higher organisms. In single-cell eukaryotes, however, caffeine somehow impairs cellular fitness by compromising cell wall integrity, inhibiting target of rapamycin (TOR) signaling and growth, and overriding cell cycle arrest caused by DNA damage. Among its multiple inhibitory targets, caffeine specifically interacts with phosphatidylinositol 3-kinase (PI3K)-related kinases causing radiosensitization and cytotoxicity via specialized intermediate molecules. Caffeine potentiates the lethality of cells in conjunction with several other stressors such as oxidants, irradiation, and various toxic compounds through largely unknown mechanisms. In this review, recent findings on caffeine effects and cellular detoxification schemes are highlighted and discussed with an emphasis on the inhibitory interactions between caffeine and its multiple targets in eukaryotic microorganisms such as budding and fission yeasts.

Functional interplay between the oxidative stress response and DNA damage checkpoint signaling for genome maintenance in aerobic organisms.

The DNA damage checkpoint signaling pathway is a highly conserved surveillance mechanism that ensures genome integrity by sequential activation of protein kinase cascades. In mammals, the main pathway is orchestrated by two central sensor kinases, ATM and ATR, that are activated in response to DNA damage and DNA replication stress. Patients lacking functional ATM or ATR suffer from ataxia-telangiectasia (A-T) or Seckel syndrome, respectively, with pleiotropic degenerative phenotypes. In addition to DNA strand breaks, ATM and ATR also respond to oxidative DNA damage and reactive oxygen species (ROS), suggesting an unconventional function as regulators of intracellular redox status. Here, we summarize the multiple roles of ATM and ATR, and of their orthologs in Saccharomyces cerevisiae, Tel1 and Mec1, in DNA damage checkpoint signaling and the oxidative stress response, and discuss emerging ideas regarding the possible mechanisms underlying the elaborate crosstalk between those pathways. This review may provide new insights into the integrated cellular strategies responsible for maintaining genome stability in eukaryotes with a focus on the yeast model organism.

The role of Rsv1 in the transcriptional regulation of genes involved in sugar metabolism for long-term survival.

Glucose limitation is a major stress condition that cells must respond to by altering their metabolism to ensure survival. Rsv1 is a zinc finger protein previously shown to be required for survival during stationary phase. In this study, we present a novel mechanism regulated by Rsv1 in the fission yeast Schizosaccharomyces pombe that is involved in altering glucose metabolic flux. We found that rsv1 gene expression is induced by Rst2 and Atf1, two transcription factors regulated by the cAMP-dependent protein kinase (PKA) pathway and the mitogen-activated protein kinase (MAPK) cascade, respectively. The downstream target genes of Rsv1 were identified by genome-wide ChIP sequencing of Rsv1-bound DNA sites and RNA sequencing analysis of Rsv1-dependent transcripts that were differentially expressed under glucose starvation. Rsv1 directly regulated the expression of at least 21 genes that mostly encode transporters and proteins related to sugar metabolism. Among these, gcd1, which encodes glucose dehydrogenase in the gluconate shunt for the pentose phosphate pathway, was most remarkably repressed by Rsv1. The defect in survival of Δrsv1 mutant under glucose starvation condition was mitigated by additional deletion of a gcd1, idn1, or a gene for a putative lactonase (SPCC16c4.10), suggesting the critical importance of downregulating the gluconate shunt and pentose phosphate pathway for long-term survival. These results show an intricate response to glucose starvation: increasing the synthesis of a transcription factor via two signal transduction pathways, which sheds light on the importance of remodeling a metabolic circuit to secure glucose for cell survival.

Synthetic lethal interaction between oxidative stress response and DNA damage repair in the budding yeast and its application to targeted anticancer therapy.

Synthetic lethality is an extreme form of negative genetic epistasis that arises when a combination of functional deficiency in two or more genes results in cell death, whereas none of the single genetic perturbations are lethal by themselves. This unconventional genetic interaction is a modification of the concept of essentiality that can be exploited for the purpose of targeted cancer therapy. The yeast Saccharomyces cerevisiae has been pivotally used for early large-scale synthetic lethal screens due to its experimental advantages, but recent advances in gene silencing technology have now made direct high-throughput analysis possible in higher organisms. Identification of tumor-specific alterations and characterization of the mechanistic principles underlying synthetic lethal interaction are the key to applying synthetic lethality to clinical cancer treatment by enabling genome-driven oncological research. Here, we provide emerging ideas on the synthetic lethal interactions in budding yeast, particularly between cellular processes responsible for oxidative stress response and DNA damage repair, and discuss how they can be appropriately utilized for context-dependent cancer therapeutics.

Lack of superoxide dismutase in a rad51 mutant exacerbates genomic instability and oxidative stress-mediated cytotoxicity in Saccharomyces cerevisiae.

A genetic analysis of synthetic lethal interactions in yeast revealed that the mutation of SOD1, encoding an antioxidant enzyme that scavenges superoxide anion radical, impaired the growth of a set of mutants defective in homologous recombination (HR) pathway. Hence, SOD1 inhibition has been proposed as a promising approach for the selective killing of HR-deficient cancer cells. However, we show that the deletion of RAD51 and SOD1 is not synthetic lethal but displays considerably slow growth and synergistic sensitivity to both reactive oxygen species (ROS)- and DNA double-strand break (DSB)-generating drugs in the budding yeast Saccharomyces cerevisiae. The function of Sod1 in regard to Rad51 is dependent on Ccs1, a copper chaperone for Sod1. Sod1 deficiency aggravates genomic instability in conjunction with the absence of Rad51 by inducing DSBs and an elevated mutation frequency. Inversely, lack of Rad51 causes a Sod1 deficiency-derived increase of intracellular ROS levels. Taken together, our results indicate that there is a significant and specific crosstalk between two major cellular damage response pathways, ROS signaling and DSB repair, for cell survival.

Unraveling new functions of superoxide dismutase using yeast model system: Beyond its conventional role in superoxide radical scavenging.

To deal with chemically reactive oxygen molecules constantly threatening aerobic life, cells are readily equipped with elaborate biological antioxidant systems. Superoxide dismutase is a metalloenzyme catalytically eliminating superoxide radical as a first-line defense mechanism against oxidative stress. Multiple different SOD isoforms have been developed throughout evolution to play distinct roles in separate subcellular compartments. SOD is not essential for viability of most aerobic organisms and intriguingly found even in strictly anaerobic bacteria. Sod1 has recently been known to play important roles as a nuclear transcription factor, an RNA binding protein, a synthetic lethal interactor, and a signal modulator in glucose metabolism, most of which are independent of its canonical function as an antioxidant enzyme. In this review, recent advances in understanding the unconventional role of Sod1 are highlighted and discussed with an emphasis on its genetic crosstalk with DNA damage repair/checkpoint pathways. The budding yeast Saccharomyces cerevisiae has been successfully used as an efficient tool and a model organism to investigate a number of novel functions of Sod1.

Anticancer Effects of the Marine Sponge Lipastrotethya sp. Extract on Wild-Type and p53 Knockout HCT116 Cells.

Interest in marine bioresources is increasing in the drug development sector. In particular, marine sponges produce a wide range of unique metabolites that enable them to survive in challenging environments, which makes them attractive sources of candidate pharmaceuticals. In previous study, we investigated over 40 marine specimens collected in Micronesia and provided by the Korean Institute of Ocean Science and Technology, for their antiproliferative effects on various cancer cell lines, and Lipastrotethya sp. extract (LSSE) was found to have a marked antiproliferative effect. In the present study, we investigated the mechanism responsible for its anticancer effect on wild-type p53 (WT) or p53 knockout (KO) HCT116 cells. LSSE inhibited cell viability and induced apoptotic cell death more so in HCT116 p53 KO cells than the WT. HCT116 WT cells treated with LSSE underwent apoptosis associated with the induction of p53 and its target genes. On the other hand, in HCT116 p53 KO cells, LSSE reduced mTOR and Bcl-2 and increased Beclin-1 and LC3-II protein levels, suggesting autophagy induction. These results indicate that the mechanisms responsible for the anticancer effect of LSSE depend on p53 status.

Yap1 and Skn7 genetically interact with Rad51 in response to oxidative stress and DNA double-strand break in Saccharomyces cerevisiae.

Reactive oxygen species (ROS)-mediated DNA adducts as well as DNA strand breaks are highly mutagenic leading to genomic instability and tumorigenesis. DNA damage repair pathways and oxidative stress response signaling have been proposed to be highly associated, but the underlying interaction remains unknown. In this study, we employed mutant strains lacking Rad51, the homolog of E. coli RecA recombinase, and Yap1 or Skn7, two major transcription factors responsive to ROS, to examine genetic interactions between double-strand break (DSB) repair proteins and cellular redox regulators in budding yeast Saccharomyces cerevisiae. Abnormal expression of YAP1 or SKN7 aggravated the mutation rate of rad51 mutants and their sensitivity to DSB- or ROS-generating reagents. Rad51 deficiency exacerbated genome instability in the presence of increased levels of ROS, and the accumulation of DSB lesions resulted in elevated intracellular ROS levels. Our findings suggest that evident crosstalk between DSB repair pathways and ROS signaling proteins contributes to cell survival and maintenance of genome integrity in response to genotoxic stress.

Mechanisms of a novel anticancer therapeutic strategy involving atmospheric pressure plasma-mediated apoptosis and DNA strand break formation.

Atmospheric pressure plasma has been developed for a variety of biomedical applications due to its chemically reactive components. Recently, the plasma has emerged as a promising novel cancer therapy based on its ability to selectively ablate cancer cells while leaving normal cells essentially unaffected. The therapeutic effect of plasma is attributed to intracellular generation of reactive oxygen/nitrogen species (ROS/RNS) leading to mitochondria-mediated apoptosis and to activation of the DNA damage checkpoint signaling pathway via severe DNA strand break formation. However, the biochemical mechanisms responsible for appropriate activation of these physiological events and which pathway is more crucial for plasma-mediated cytotoxicity have not been clarified. Understanding the molecular link between ROS/RNS-mediated apoptosis and DNA damage-involved chromosome instability is critical for the development of more efficacious therapeutic strategies for selective killing of diverse cancer cells.

Translesion Polymerases Drive Microhomology-Mediated Break-Induced Replication Leading to Complex Chromosomal Rearrangements.

Complex genomic rearrangements (CGRs) are a hallmark of many human diseases. Recently, CGRs were suggested to result from microhomology-mediated break-induced replication (MMBIR), a replicative mechanism involving template switching at positions of microhomology. Currently, the cause of MMBIR and the proteins mediating this process remain unknown. Here, we demonstrate in yeast that a collapse of homology-driven break-induced replication (BIR) caused by defective repair DNA synthesis in the absence of Pif1 helicase leads to template switches involving 0-6 nt of homology, followed by resolution of recombination intermediates into chromosomal rearrangements. Importantly, we show that these microhomology-mediated template switches, indicative of MMBIR, are driven by translesion synthesis (TLS) polymerases Polζ and Rev1. Thus, an interruption of BIR involving fully homologous chromosomes in yeast triggers a switch to MMBIR catalyzed by TLS polymerases. Overall, our study provides important mechanistic insights into the initiation of MMBIR associated with genomic rearrangements, similar to those promoting diseases in humans.

Atmospheric-pressure plasma jet induces DNA double-strand breaks that require a Rad51-mediated homologous recombination for repair in Saccharomyces cerevisiae.

Non-thermal plasma generated under atmospheric pressure produces a mixture of chemically reactive molecules and has been developed for a number of biomedical applications. Recently, plasma jet has been proposed as novel cancer therapies based on the observation that free radicals generated by plasma jet induce mitochondria-mediated apoptotic cell death. We show here that air plasma jet induces DNA double-strand breaks (DSBs) in yeast chromosomes leading to genomic instability and loss of viability, which are alleviated by Rad51, the yeast homolog of Escherichiacoli RecA recombinase, through DNA damage repair by a homologous recombination (HR) process. Hypersensitivity of rad51 mutant to air plasma was not restored by antioxidant treatment unlike sod1 mutant that was highly sensitive to reactive oxygen species (ROS) challenge, suggesting that plasma jet induces DSB-mediated cell death independent of ROS generation. These results may provide a new insight into the mechanism of air plasma jet-induced cell death.

To peep into Pif1 helicase: multifaceted all the way from genome stability to repair-associated DNA synthesis.

Pif1 DNA helicase is the prototypical member of a 5' to 3' helicase superfamily conserved from bacteria to humans. In Saccharomyces cerevisiae, Pif1 and its homologue Rrm3, localize in both mitochondria and nucleus playing multiple roles in the maintenance of genomic homeostasis. They display relatively weak processivities in vitro, but have largely non-overlapping functions on common genomic loci such as mitochondrial DNA, telomeric ends, and many replication forks especially at hard-to-replicate regions including ribosomal DNA and G-quadruplex structures. Recently, emerging evidence shows that Pif1, but not Rrm3, has a significant new role in repair-associated DNA synthesis with Polδ during homologous recombination stimulating D-loop migration for conservative DNA replication. Comparative genetic and biochemical studies on the structure and function of Pif1 family helicases across different biological systems are further needed to elucidate both diversity and specificity of their mechanisms of action that contribute to genome stability.

Pif1 helicase and Polδ promote recombination-coupled DNA synthesis via bubble migration.

During DNA repair by homologous recombination (HR), DNA synthesis copies information from a template DNA molecule. Multiple DNA polymerases have been implicated in repair-specific DNA synthesis, but it has remained unclear whether a DNA helicase is involved in this reaction. A good candidate DNA helicase is Pif1, an evolutionarily conserved helicase in Saccharomyces cerevisiae important for break-induced replication (BIR) as well as HR-dependent telomere maintenance in the absence of telomerase found in 10-15% of all cancers. Pif1 has a role in DNA synthesis across hard-to-replicate sites and in lagging-strand synthesis with polymerase δ (Polδ). Here we provide evidence that Pif1 stimulates DNA synthesis during BIR and crossover recombination. The initial steps of BIR occur normally in Pif1-deficient cells, but Polδ recruitment and DNA synthesis are decreased, resulting in premature resolution of DNA intermediates into half-crossovers. Purified Pif1 protein strongly stimulates Polδ-mediated DNA synthesis from a D-loop made by the Rad51 recombinase. Notably, Pif1 liberates the newly synthesized strand to prevent the accumulation of topological constraint and to facilitate extensive DNA synthesis via the establishment of a migrating D-loop structure. Our results uncover a novel function of Pif1 and provide insights into the mechanism of HR.

Cell cycle regulation of DNA double-strand break end resection by Cdk1-dependent Dna2 phosphorylation.

DNA recombination pathways are regulated by the cell cycle to coordinate with replication. Cyclin-dependent kinase (Cdk1) promotes efficient 5' strand resection at DNA double-strand breaks (DSBs), the initial step of homologous recombination and damage checkpoint activation. The Mre11-Rad50-Xrs2 complex with Sae2 initiates resection, whereas two nucleases, Exo1 and Dna2, and the DNA helicase-topoisomerase complex Sgs1-Top3-Rmi1 generate longer ssDNA at DSBs. Using Saccharomyces cerevisiae, we provide evidence for Cdk1-dependent phosphorylation of the resection nuclease Dna2 at Thr4, Ser17 and Ser237 that stimulates its recruitment to DSBs, resection and subsequent Mec1-dependent phosphorylation. Poorly recruited dna2T4A S17A S237A and dna2ΔN248 mutant proteins promote resection only in the presence of Exo1, suggesting cross-talk between Dna2- and Exo1-dependent resection pathways.

Leptin enhances nitric oxide-dependent relaxation of the clitoral corpus cavernosum.

PURPOSE: The effects of leptin on female sexual behaviors are controversial, and studies on this topic are limited. The objectives of this study were to evaluate the direct effects of leptin on clitoral vasoreactivity in vitro and to determine the mechanism of action. MATERIALS AND METHODS: Isometric tension studies were conducted to determine the effects of pretreatment with leptin (10(-8) M) on the contractile responses of rabbit clitoral corpus cavernosal smooth muscle strips. The effects of leptin were assessed on precontraction induced by phenylephrine (PE; 10(-9)-10(-4) M) and KCl (35-140 mM). We also examined the effect of leptin on relaxation induced by acetylcholine (ACh; 10(-9)-10(-4) M), verapamil (10(-10)-10(-6) M), and sodium nitroprusside (10(-9)-10(-4) M) in PE-precontracted (10(-5) M) strips. RESULTS: Leptin enhanced ACh-induced relaxation in PE-precontracted strips. L-NAME pretreatment significantly reduced the effect of leptin on ACh-induced relaxation, whereas L-arginine potentiated the effect of leptin. Leptin decreased the KCl-induced contractile responses. Leptin increased verapamil-induced relaxation responses. The relaxation effects of leptin on KCl-induced contraction were inhibited by 10(-5) M methylene blue and L-NAME pretreatment. CONCLUSIONS: A high concentration of leptin enhances ACh-dependent relaxation in clitoral cavernosal smooth muscles. These relaxation effects of leptin may occur through an NO-dependent mechanism and voltage-dependent calcium channel blockade.

Saccharomyces cerevisiae Mre11/Rad50/Xrs2 and Ku proteins regulate association of Exo1 and Dna2 with DNA breaks.

Single-stranded DNA constitutes an important early intermediate for homologous recombination and damage-induced cell cycle checkpoint activation. In Saccharomyces cerevisiae, efficient double-strand break (DSB) end resection requires several enzymes; Mre11/Rad50/Xrs2 (MRX) and Sae2 are implicated in the onset of 5'-strand resection, whereas Sgs1/Top3/Rmi1 with Dna2 and Exo1 are involved in extensive resection. However, the molecular events leading to a switch from the MRX/Sae2-dependent initiation to the Exo1- and Dna2-dependent resection remain unclear. Here, we show that MRX recruits Dna2 nuclease to DSB ends. MRX also stimulates recruitment of Exo1 and antagonizes excess binding of the Ku complex to DSB ends. Using resection assay with purified enzymes in vitro, we found that Ku and MRX regulate the nuclease activity of Exo1 in an opposite way. Efficient loading of Dna2 and Exo1 requires neither Sae2 nor Mre11 nuclease activities. However, Mre11 nuclease activity is essential for resection in the absence of extensive resection enzymes. The results provide new insights into how MRX catalyses end resection and recombination initiation.

Mechanism of the ATP-dependent DNA end-resection machinery from Saccharomyces cerevisiae.

If not properly processed and repaired, DNA double-strand breaks (DSBs) can give rise to deleterious chromosome rearrangements, which could ultimately lead to the tumour phenotype. DSB ends are resected in a 5' to 3' fashion in cells, to yield single-stranded DNA (ssDNA) for the recruitment of factors critical for DNA damage checkpoint activation and repair by homologous recombination. The resection process involves redundant pathways consisting of nucleases, DNA helicases and associated proteins. Being guided by recent genetic studies, we have reconstituted the first eukaryotic ATP-dependent DNA end-resection machinery comprising the Saccharomyces cerevisiae Mre11-Rad50-Xrs2 (MRX) complex, the Sgs1-Top3-Rmi1 complex, Dna2 protein and the heterotrimeric ssDNA-binding protein RPA. Here we show that DNA strand separation during end resection is mediated by the Sgs1 helicase function, in a manner that is enhanced by Top3-Rmi1 and MRX. In congruence with genetic observations, although the Dna2 nuclease activity is critical for resection, the Mre11 nuclease activity is dispensable. By examining the top3 Y356F allele and its encoded protein, we provide evidence that the topoisomerase activity of Top3, although critical for the suppression of crossover recombination, is not needed for resection either in cells or in the reconstituted system. Our results also unveil a multifaceted role of RPA, in the sequestration of ssDNA generated by DNA unwinding, enhancement of 5' strand incision, and protection of the 3' strand. Our reconstituted system should serve as a useful model for delineating the mechanistic intricacy of the DNA break resection process in eukaryotes.