Interaction of Proteins with Inverted Repeats and Cruciform Structures in Nucleic Acids.

Cruciforms occur when inverted repeat sequences in double-stranded DNA adopt intra-strand hairpins on opposing strands. Biophysical and molecular studies of these structures confirm their characterization as four-way junctions and have demonstrated that several factors influence their stability, including overall chromatin structure and DNA supercoiling. Here, we review our understanding of processes that influence the formation and stability of cruciforms in genomes, covering the range of sequences shown to have biological significance. It is challenging to accurately sequence repetitive DNA sequences, but recent advances in sequencing methods have deepened understanding about the amounts of inverted repeats in genomes from all forms of life. We highlight that, in the majority of genomes, inverted repeats are present in higher numbers than is expected from a random occurrence. It is, therefore, becoming clear that inverted repeats play important roles in regulating many aspects of DNA metabolism, including replication, gene expression, and recombination. Cruciforms are targets for many architectural and regulatory proteins, including topoisomerases, p53, Rif1, and others. Notably, some of these proteins can induce the formation of cruciform structures when they bind to DNA. Inverted repeat sequences also influence the evolution of genomes, and growing evidence highlights their significance in several human diseases, suggesting that the inverted repeat sequences and/or DNA cruciforms could be useful therapeutic targets in some cases.

Impacts of Molecular Structure on Nucleic Acid-Protein Interactions.

Interactions between nucleic acids and proteins are some of the most important interactions in biology because they are the cornerstones for fundamental biological processes, such as replication, transcription, and recombination [...].

New telomere to telomere assembly of human chromosome 8 reveals a previous underestimation of G-quadruplex forming sequences and inverted repeats.

Taking advantage of evolving and improving sequencing methods, human chromosome 8 is now available as a gapless, end-to-end assembly. Thanks to advances in long-read sequencing technologies, its centromere, telomeres, duplicated gene families and repeat-rich regions are now fully sequenced. We were interested to assess if the new assembly altered our understanding of the potential impact of non-B DNA structures within this completed chromosome sequence. It has been shown that non-B secondary structures, such as G-quadruplexes, hairpins and cruciforms, have important regulatory functions and potential as targeted therapeutics. Therefore, we analysed the presence of putative G-quadruplex forming sequences and inverted repeats in the current human reference genome (GRCh38) and in the new end-to-end assembly of chromosome 8. The comparison revealed that the new assembly contains significantly more inverted repeats and G-quadruplex forming sequences compared to the current reference sequence. This observation can be explained by improved accuracy of the new sequencing methods, particularly in regions that contain extensive repeats of bases, as is preferred by many non-B DNA structures. These results show a significant underestimation of the prevalence of non-B DNA secondary structure in previous assembly versions of the human genome and point to their importance being not fully appreciated. We anticipate that similar observations will occur as the improved sequencing technologies fill in gaps across the genomes of humans and other organisms.

GSH Levels Serve As a Biological Redox Switch Regulating Sulforaphane-Induced Cell Fate in Human Lens Cells.

Purpose: Sulforaphane (SFN) is a therapeutic phytochemical agent for many health conditions. SFN-induced cytotoxicity is shown to have promise in preventing posterior capsule opacification (PCO). In the current study, we aimed to elucidate key processes and mechanisms linking SFN treatment to lens cell death. Methods: The human lens epithelial cell line FHL124 and central anterior epithelium were used as experimental models. Cell death was assessed by microscopic observation and cell damage/viability assays. Gene or protein levels were assessed by TaqMan RT-PCR or immunoblotting. Mitochondrial networks and DNA damage were assessed by immunofluorescence. Mitochondrial membrane potential, activating transcription factor 6 (ATF6) activity, ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG), and glutathione reductase (GR) activity were measured using different light reporter assays. SFN metabolites were analyzed by LC-MS/MS. Results: Treatment with N-acetylcysteine (NAC), a reactive oxygen species scavenger, prevented SFN-induced cell death in both models. NAC also significantly protected FHL124 cells from SFN-induced mitochondrial dysfunctions, endoplasmic reticulum stress (ERS), DNA damage and autophagy. SFN significantly depleted GSH, the major antioxidant in the eye, and reduced GR activity, despite doubling its protein levels. The most abundant SFN conjugate detected in lens cells following SFN application was SFN-GSH. The addition of GSH protected lens cells from all SFN-induced cellular events. Conclusions: SFN depletes GSH levels in lens cells through conjugation and inhibition of GR activity. This leads to increased reactive oxygen species and oxidative stress that trigger mitochondrial dysfunction, ERS, autophagy, and DNA damage, leading to cell death. In summary, the work presented provides a mechanistic understanding to support the therapeutic application of SFN for PCO and other disorders.

Evolution of Diverse Strategies for Promoter Regulation.

DNA is fundamentally important for all cellular organisms due to its role as a store of hereditary genetic information. The precise and accurate regulation of gene transcription depends primarily on promoters, which vary significantly within and between genomes. Some promoters are rich in specific types of bases, while others have more varied, complex sequence characteristics. However, it is not only base sequence but also epigenetic modifications and altered DNA structure that regulate promoter activity. Significantly, many promoters across all organisms contain sequences that can form intrastrand hairpins (cruciforms) or four-stranded structures (G-quadruplex or i-motif). In this review we integrate recent studies on promoter regulation that highlight the importance of DNA structure in the evolutionary adaptation of promoter sequences.

Structures and stability of simple DNA repeats from bacteria.

DNA is a fundamentally important molecule for all cellular organisms due to its biological role as the store of hereditary, genetic information. On the one hand, genomic DNA is very stable, both in chemical and biological contexts, and this assists its genetic functions. On the other hand, it is also a dynamic molecule, and constant changes in its structure and sequence drive many biological processes, including adaptation and evolution of organisms. DNA genomes contain significant amounts of repetitive sequences, which have divergent functions in the complex processes that involve DNA, including replication, recombination, repair, and transcription. Through their involvement in these processes, repetitive DNA sequences influence the genetic instability and evolution of DNA molecules and they are located non-randomly in all genomes. Mechanisms that influence such genetic instability have been studied in many organisms, including within human genomes where they are linked to various human diseases. Here, we review our understanding of short, simple DNA repeats across a diverse range of bacteria, comparing the prevalence of repetitive DNA sequences in different genomes. We describe the range of DNA structures that have been observed in such repeats, focusing on their propensity to form local, non-B-DNA structures. Finally, we discuss the biological significance of such unusual DNA structures and relate this to studies where the impacts of DNA metabolism on genetic stability are linked to human diseases. Overall, we show that simple DNA repeats in bacteria serve as excellent and tractable experimental models for biochemical studies of their cellular functions and influences.

p53 Binds Preferentially to Non-B DNA Structures Formed by the Pyrimidine-Rich Strands of GAA·TTC Trinucleotide Repeats Associated with Friedreich's Ataxia.

Expansions of trinucleotide repeats (TNRs) are associated with genetic disorders such as Friedreich's ataxia. The tumor suppressor p53 is a central regulator of cell fate in response to different types of insults. Sequence and structure-selective modes of DNA recognition are among the main attributes of p53 protein. The focus of this work was analysis of the p53 structure-selective recognition of TNRs associated with human neurodegenerative diseases. Here, we studied binding of full length p53 and several deletion variants to TNRs folded into DNA hairpins or loops. We demonstrate that p53 binds to all studied non-B DNA structures, with a preference for non-B DNA structures formed by pyrimidine (Py) rich strands. Using deletion mutants, we determined the C-terminal DNA binding domain of p53 to be crucial for recognition of such non-B DNA structures. We also observed that p53 in vitro prefers binding to the Py-rich strand over the purine (Pu) rich strand in non-B DNA substrates formed by sequence derived from the first intron of the frataxin gene. The binding of p53 to this region was confirmed using chromatin immunoprecipitation in human Friedreich's ataxia fibroblast and adenocarcinoma cells. Altogether these observations provide further evidence that p53 binds to TNRs' non-B DNA structures.

Antioxidant effects of sulforaphane in human HepG2 cells and immortalised hepatocytes.

Sulforaphane (SFN) has shown anti-cancer effects in cellular and animal studies but its effectiveness has been limited in human studies. Here, the effects of SFN were measured in both human hepatocytes (HHL5) and hepatoma (HepG2) cells. Results showed that SFN inhibited cell viability and induced DNA strand breaks at high doses (≥20 µM). It also activated the nuclear factor (erythroid-derived 2)-like 2 (Nrf2), and increased intracellular glutathione (GSH) levels at 24 h. Pre-treatment with a low dose SFN (≤5 µM) protected against hydrogen peroxide (H2O2)-induced cell damage. High doses of SFN were more toxic towards HHL5 compared to HepG2 cells; the difference is likely due to the disparity in the responses of Nrf2-driven enzymes and -GSH levels between the two cell lines. In addition, HepG2 cells hijacked the cytoprotective effect of SFN over a wider dose range (1.25-20 µM) compared to HHL5. Manipulation of levels of GSH and Nrf2 in HepG2 cells confirmed that both molecules mediate the protective effects of SFN against H2O2. The non-specific nature of SFN in the regulation of cell death and survival could present undesirable risks, i.e. be more toxic to normal cells, and cause chemo-resistance in tumor cells. These issues should be addressed in the context for cancer prevention and treatment before large scale clinical trials are undertaken.

Chemopreventive Activities of Sulforaphane and Its Metabolites in Human Hepatoma HepG2 Cells.

Sulforaphane (SFN) exhibits chemopreventive effects through various mechanisms. However, few studies have focused on the bioactivities of its metabolites. Here, three metabolites derived from SFN were studied, known as sulforaphane glutathione, sulforaphane cysteine and sulforaphane-N-acetylcysteine. Their effects on cell viability, DNA damage, tumorigenicity, cell migration and adhesion were measured in human hepatoma HepG2 cells, and their anti-angiogenetic effects were determined in a 3D co-culture model of human umbilical vein endothelial cells (HUVECs) and pericytes. Results indicated that these metabolites at high doses decreased cancer cell viability, induced DNA damage and inhibited motility, and impaired endothelial cell migration and tube formation. Additionally, pre-treatment with low doses of SFN metabolites protected against H2O2 challenge. The activation of the nuclear factor E2-related factor 2 (Nrf2)-antioxidant response element (ARE) pathway and the induction of intracellular glutathione (GSH) played an important role in the cytoprotective effects of SFN metabolites. In conclusion, SFN metabolites exhibited similar cytotoxic and cytoprotective effects to SFN, which proves the necessity to study the mechanisms of action of not only SFN but also of its metabolites. Based on the different tissue distribution profiles of these metabolites, the most relevant chemical forms can be selected for targeted chemoprevention.

Sulforaphane promotes ER stress, autophagy, and cell death: implications for cataract surgery.

Posterior capsule opacification (PCO) commonly develops following cataract surgery and is a wound-healing response that can ultimately lead to secondary visual loss. Improved management of this problem is required. The isothiocyanate, sulforaphane (SFN), is reported to exert cytoprotective and cytotoxic actions, and the latter may be exploited to treat/prevent PCO. SFN concentrations of 10 µM and above significantly impaired wound-healing in a human lens capsular bag model. A similar pattern of response was also seen with a human lens cell line, FHL124. SFN treatment promoted increased expression of endoplasmic reticulum (ER) stress genes, which also corresponded with protein expression. Evidence of autophagy was observed in response to SFN as determined by increased microtubule-associated protein 1A/1B-light chain 3 (LC3)-II levels and detection of autophagic vesicles. This response was disrupted by established autophagy inhibitors chloroquine and 3-MA. SFN was found to promote MAPK signaling, and inhibition of ERK activation using U0126 prevented SFN-induced LC3-II elevation and vesicle formation. SFN also significantly increased levels of reactive oxygen species. Taken together, our findings suggest that SFN is capable of reducing lens cell growth and viability and thus could serve as a putative therapeutic agent for PCO. KEY MESSAGE: SFN reduces lens epithelial cell growth, migration, and viability. SFN can promote ER stress and autophagy in lens cells. SFN promotes MAPK signaling, and inhibition of MEK can suppress SFN-induced autophagy. ER stress and autophagy in lens cells are likely promoted by ROS production. SFN may help prevent posterior capsule opacification after cataract surgery.

Prelamin A impairs 53BP1 nuclear entry by mislocalizing NUP153 and disrupting the Ran gradient.

The nuclear lamina is essential for the proper structure and organization of the nucleus. Deregulation of A-type lamins can compromise genomic stability, alter chromatin organization and cause premature vascular aging. Here, we show that accumulation of the lamin A precursor, prelamin A, inhibits 53BP1 recruitment to sites of DNA damage and increases basal levels of DNA damage in aged vascular smooth muscle cells. We identify that this genome instability arises through defective nuclear import of 53BP1 as a consequence of abnormal topological arrangement of nucleoporin NUP153. We show for the first time that this nucleoporin is important for the nuclear localization of Ran and that the deregulated Ran gradient is likely to be compromising the nuclear import of 53BP1. Importantly, many of the defects associated with prelamin A expression were significantly reduced upon treatment with Remodelin, a small molecule recently reported to reverse deficiencies associated with abnormal nuclear lamina.

PARP-1 inhibition influences the oxidative stress response of the human lens.

Poly(ADP-ribose) polymerase-1 (PARP-1) is best characterised for its involvement in DNA repair. PARP-1 activity is also linked to cell fate, confounding its roles in maintaining genome integrity. The current study assessed the functional roles of PARP-1 within human lens cells in response to oxidative stress. The human lens epithelial cell line FHL124 and whole human lens cultures were used as experimental systems. Hydrogen peroxide (H2O2) was employed to induce oxidative stress and cell death was assessed by LDH release. The functional influence of PARP-1 was assessed using targeted siRNA and chemical inhibition (by AG14361). Immunocytochemistry and western blotting were used to assess PARP-1 expression and the alkaline comet assay determined the levels of DNA strand breaks. PARP-1 was generally observed in the cell nucleus in both the FHL124 cell line and whole human lenses. PARP-1 inhibition rendered FHL124 cells more susceptible to H2O2-induced DNA strand breaks. Interestingly, reduction of PARP-1 activity significantly inhibited H2O2-induced cell death relative to control cells. Inhibition of PARP-1 in whole human lenses resulted in a reduced level of lens opacity and cell death following exposure to H2O2 relative to matched pair controls. Thus, we show that PARP-1 could play a role in the fate of human lens cells, and these first observations in human lenses suggest that it could impact on lens opacity. Further studies are required to elucidate the regulatory processes that give rise to these effects.

Promoting microbiology education through the iGEM synthetic biology competition.

Synthetic biology has developed rapidly in the 21st century. It covers a range of scientific disciplines that incorporate principles from engineering to take advantage of and improve biological systems, often applied to specific problems. Methods important in this subject area include the systematic design and testing of biological systems and, here, we describe how synthetic biology projects frequently develop microbiology skills and education. Synthetic biology research has huge potential in biotechnology and medicine, which brings important ethical and moral issues to address, offering learning opportunities about the wider impact of microbiological research. Synthetic biology projects have developed into wide-ranging training and educational experiences through iGEM, the International Genetically Engineered Machines competition. Elements of the competition are judged against specific criteria and teams can win medals and prizes across several categories. Collaboration is an important element of iGEM, and all DNA constructs synthesized by iGEM teams are made available to all researchers through the Registry for Standard Biological Parts. An overview of microbiological developments in the iGEM competition is provided. This review is targeted at educators that focus on microbiology and synthetic biology, but will also be of value to undergraduate and postgraduate students with an interest in this exciting subject area.

Base-modified NAD and AMP derivatives and their activity against bacterial DNA ligases.

We report the chemical synthesis and conformational analysis of a collection of 2-, 6- and 8-substituted derivatives of ß-NAD(+) and AMP, and their biochemical evaluation against NAD(+)-dependent DNA ligases from Escherichia coli and Mycobacterium tuberculosis. Bacterial DNA ligases are validated anti-microbial targets, and new strategies for their inhibition are therefore of considerable scientific and practical interest. Our study includes several pairs of ß-NAD(+) and AMP derivatives with the same substitution pattern at the adenine base. This has enabled the first direct comparison of co-substrate and inhibitor behaviour against bacterial DNA ligases. Our results suggest that an additional substituent in position 6 or 8 of the adenine base in ß-NAD(+) is detrimental for activity as either co-substrate or inhibitor. In contrast, substituents in position 2 are not only tolerated, but appear to give rise to a new mode of inhibition, which targets the conformational changes these DNA ligases undergo during catalysis. Using a molecular modelling approach, we highlight that these findings have important implications for our understanding of ligase mechanism and inhibition, and may provide a promising starting point for the rational design of a new class of inhibitors against NAD(+)-dependent DNA ligases.

Enzyme-linked electrochemical DNA ligation assay using magnetic beads.

DNA ligases are essential enzymes in all cells and have been proposed as targets for novel antibiotics. Efficient DNA ligase activity assays are thus required for applications in biomedical research. Here we present an enzyme-linked electrochemical assay based on two terminally tagged probes forming a nicked junction upon hybridization with a template DNA. Nicked DNA bearing a 5' biotin tag is immobilized on the surface of streptavidin-coated magnetic beads, and ligated product is detected via a 3' digoxigenin tag recognized by monoclonal antibody-alkaline phosphatase conjugate. Enzymatic conversion of napht-1-yl phosphate to napht-1-ol enables sensitive detection of the voltammetric signal on a pyrolytic graphite electrode. The technique was tested under optimal conditions and various situations limiting or precluding the ligation reaction (such as DNA substrates lacking 5'-phosphate or containing a base mismatch at the nick junction, or application of incompatible cofactor), and utilized for the analysis of the nick-joining activity of a range of recombinant Escherichia coli DNA ligase constructs. The novel technique provides a fast, versatile, specific, and sensitive electrochemical assay of DNA ligase activity.

Sequence-specific and DNA structure-dependent interactions of Escherichia coli MutS and human p53 with DNA.

Many proteins involved in DNA repair systems interact with DNA that has structure altered from the typical B-form helix. Using magnetic beads to immobilize DNAs containing various types of structures, we evaluated the in vitro binding activities of two well-characterized DNA repair proteins, Escherichia coli MutS and human p53. E. coli MutS bound to double-stranded DNAs, with higher affinity for a G/T mismatch compared to a G/A mismatch and highest affinity for larger non-B-DNA structures. E. coli MutS bound best to DNA between pH 6 and 9. Experiments discriminated between modes of p53-DNA binding, and increasing ionic strength reduced p53 binding to nonspecific double-stranded DNA, but had minor effects on binding to consensus response sequences or single-stranded DNA. Compared to nonspecific DNA sequences, p53 bound with a higher affinity to mismatches and base insertions, while binding to various hairpin structures was similar to that observed to its consensus DNA sequence. For hairpins containing CTG repeats, the extent of p53 binding was proportional to the size of the repeat. In summary, using the flexibility of the magnetic bead separation assay we demonstrate that pH and ionic strength influence the binding of two DNA repair proteins to a variety of DNA structures.

Sulforaphane can protect lens cells against oxidative stress: implications for cataract prevention.

PURPOSE: Protecting the lens against oxidative stress is of great importance in delaying the onset of cataract. Isothiocyanates, such as sulforaphane (SFN), are proposed to provide cytoprotection against oxidative stress. We therefore tested the ability of SFN to perform this role in lens cells and establish its ability to delay the onset of cataract. METHODS: The human lens epithelial cell line FHL124 and whole porcine lens culture systems were used. The ApoTox-Glo Triplex Assay was used to assess FHL124 cell survival, cytotoxicity, and apoptosis. The MTS assay was used to assess cell populations. To determine levels of DNA strand breaks, the alkaline comet assay was performed and quantified. Lactate dehydrogenase levels in the medium were evaluated to reflect cell damage/death. To assess level of gene expression, an Illumina whole-genome HT-12 v4 beadchip was used. Protein expression was determined by Western blot and immunocytochemistry. RESULTS: Exposures of 30 µM H2O2 to FHL124 cells caused a reduction in cell viability and increased cytotoxicity/apoptosis; these effects were significantly inhibited by 24-hour pretreatment with 1 µM SFN. In addition, 1 µM SFN significantly reduced H2O2-induced DNA strand breaks. When applied to cultured porcine lenses, SFN protected against H2O2-induced opacification. Illumina whole-genome HT-12 v4 beadchip microarray data revealed eight genes upregulated following 24-hour exposure to 1- and 2-µM SFN, which included NQO1 and TXNRD1. This pattern was confirmed at the protein level. Nrf2 translocated to the nucleus in response to 0.5- to 2.0-µM SFN exposure CONCLUSIONS: The dietary component SFN demonstrates an ability to protect human lens cells against oxidative stress and thus could potentially delay the onset of cataract.

Sigma 1 receptor stimulation protects against oxidative damage through suppression of the ER stress responses in the human lens.

Stimulation of sigma-1 receptors is reported to protect against oxidative stress. The present study uses cells and tissue from the human lens to elucidate the relationship between the sigma 1 receptor, ER stress and oxidative stress-induced damage. Exposure of the human lens cell line FHL124 to increasing concentrations of H(2)O(2) led to reduced cell viability and increased apoptosis. In response to 30 µM H(2)O(2), levels of the ER stress proteins BiP, ATF6 and pEIF2α were significantly increased within 4h of exposure. Expression of the sigma 1 receptor was markedly increased in response to H(2)O(2). Application of 10 and 30 µM (+)-pentazocine, a sigma 1 receptor agonist, significantly inhibited the H(2)O(2) induced cell death. (+)-Pentazocine also suppressed the oxidative stress induced reduction of pro-caspase 12 and suppressed the induction of the ER stress proteins BiP and EIF2α. When applied to cultured human lenses, (+)-pentazocine protected against apoptotic cell death, LDH release and against H(2)O(2) induced opacification. These data demonstrate that stimulation of the sigma 1 receptor provides significant protection from oxidative damage and is, therefore, a putative therapeutic approach to delay the onset of diseases that may be triggered by oxidative damage, including cataract formation.

From beads on a string to the pearls of regulation: the structure and dynamics of chromatin.

The assembly of eukaryotic chromatin, and the bearing of its structural organization on the regulation of gene expression, were the central topics of a recent conference organized jointly by the Biochemical Society and Wellcome Trust. A range of talks and poster presentations covered topical aspects of this research field and illuminated recent advances in our understanding of the structure and function of chromatin. The two-day meeting had stimulating presentations complemented with lively discourse and interactions of participants. In the present paper, we summarize the topics presented at the meeting, in particular highlighting subjects that are reviewed in more detail within this issue of Biochemical Society Transactions. The reports bring to life the truly fascinating molecular and structural biology of chromatin.