Genome-wide studies of DNA replication origins revealed that origins preferentially associate with an Origin G-rich Repeated Element (OGRE), potentially forming G-quadruplexes (G4). Here, we ...functionally address their requirements for DNA replication initiation in a series of independent approaches. Deletion of the OGRE/G4 sequence strongly decreased the corresponding origin activity. Conversely, the insertion of an OGRE/G4 element created a new replication origin. This element also promoted replication of episomal EBV vectors lacking the viral origin, but not if the OGRE/G4 sequence was deleted. A potent G4 ligand, PhenDC3, stabilized G4s but did not alter the global origin activity. However, a set of new, G4-associated origins was created, whereas suppressed origins were largely G4-free. In vitro Xenopus laevis replication systems showed that OGRE/G4 sequences are involved in the activation of DNA replication, but not in the pre-replication complex formation. Altogether, these results converge to the functional importance of OGRE/G4 elements in DNA replication initiation.
We compared here 80 different sequences containing four tracts of three guanines with loops of variable length (between 1 and 15 bases for unmodified sequences, up to 30 for fluorescently labeled ...oligonucleotides). All sequences were capable of forming stable quadruplexes, with Tm above physiological temperature in most cases. Unsurprisingly, the melting temperature was systematically lower in sodium than in potassium but the difference between both ionic conditions varied between 1 and >39°C (average difference: 18.3°C). Depending on the sequence context, and especially for G4 sequences involving two very short loops, the third one may be very long without compromising the stability of the quadruplex. A strong inverse correlation between total loop length and Tm was found in K⁺: each added base leads to a 2°C drop in Tm or ~0.3 kcal/mol loss in ΔG°. The trend was less clear in Na⁺, with a longer than expected optimal loop length (up to 5 nt). This study will therefore extend the sequence repertoire of quadruplex-prone sequences, arguing for a modification of the widely used consensus (maximal loop size of 7 bases).
Thioflavin T (ThT) becomes fluorescent in the presence of the G-quadruplex structure such as that formed by the human telomeric motif. In this report, we extend and generalize these observations and ...show that this dye may be used as a convenient and specific quadruplex probe. In the presence of most, but not all, G4-forming sequences, we observed a large increase in ThT fluorescence emission, whereas the presence of control duplexes and single strands had a more limited effect on emission. This differential behavior allowed us to design a high-throughput assay to detect G4 formation. Hundreds of different oligonucleotides may be tested in parallel for G4 formation with a simple fluorescence plate reader. We applied this technique to a family of aptamers not previously recognized as G4-forming sequences and demonstrated that ThT fluorescence signal may be used to predict G4 formation.
Abstract
Guanine-rich nucleic acid sequences can fold into four-stranded G-quadruplex (G4) structures. Despite growing evidence for their biological significance, considerable work still needs to be ...done to detail their cellular occurrence and functions. Herein, we describe an optimized core-extended naphthalene diimide (cex-NDI) to be exploited as a G4 light-up sensor. The sensing mechanism relies on the shift of the aggregate-monomer equilibrium towards the bright monomeric state upon G4 binding. In contrast with the majority of other ligands, this novel cex-NDI is able to discriminate among G4s with different topologies, with a remarkable fluorescent response for the parallel ones. We investigate this sensing by means of biophysical methods, comparing the lead compound to a non-selective analogue. We demonstrate that mitigating the affinity of the binding core for G4s results in an increased selectivity and sensitivity of the fluorescent response. This is achieved by replacing positively charged substituents with diethylene glycol (DEG) side chains. Remarkably, the limit of detection values obtained for parallel G4s are more than one order of magnitude lower than those of the parallel-selective ligand N-methyl mesoporphyrin IX (NMM). Interestingly, the classical fluorescent intercalator displacement (FID) assay failed to reveal binding of cex-NDI to G4 because of the presence a ternary complex (G4-TO-cex-NDI) revealed by electrospray-MS. Our study thus provides a rational basis to design or modify existent scaffolds to redirect the binding preference of G4 ligands.
Abstract
G-quadruplex ligands exert their antiproliferative effects through telomere-dependent and telomere-independent mechanisms, but the inter-relationships among autophagy, cell growth arrest and ...cell death induced by these ligands remain largely unexplored. Here, we demonstrate that the G-quadruplex ligand 20A causes growth arrest of cancer cells in culture and in a HeLa cell xenografted mouse model. This response is associated with the induction of senescence and apoptosis. Transcriptomic analysis of 20A treated cells reveals a significant functional enrichment of biological pathways related to growth arrest, DNA damage response and the lysosomal pathway. 20A elicits global DNA damage but not telomeric damage and activates the ATM and autophagy pathways. Loss of ATM following 20A treatment inhibits both autophagy and senescence and sensitizes cells to death. Moreover, disruption of autophagy by deletion of two essential autophagy genes ATG5 and ATG7 leads to failure of CHK1 activation by 20A and subsequently increased cell death. Our results, therefore, identify the activation of ATM by 20A as a critical player in the balance between senescence and apoptosis and autophagy as one of the key mediators of such regulation. Thus, targeting the ATM/autophagy pathway might be a promising strategy to achieve the maximal anticancer effect of this compound.
In budding yeast, the Pif1 DNA helicase is involved in the maintenance of both nuclear and mitochondrial genomes, but its role in these processes is still poorly understood. Here, we provide evidence ...for a new Pif1 function by demonstrating that its absence promotes genetic instability of alleles of the G-rich human minisatellite CEB1 inserted in the Saccharomyces cerevisiae genome, but not of other tandem repeats. Inactivation of other DNA helicases, including Sgs1, had no effect on CEB1 stability. In vitro, we show that CEB1 repeats formed stable G-quadruplex (G4) secondary structures and the Pif1 protein unwinds these structures more efficiently than regular B-DNA. Finally, synthetic CEB1 arrays in which we mutated the potential G4-forming sequences were no longer destabilized in pif1Delta cells. Hence, we conclude that CEB1 instability in pif1Delta cells depends on the potential to form G-quadruplex structures, suggesting that Pif1 could play a role in the metabolism of G4-forming sequences.
G-quadruplexes (G4) are DNA secondary structures that take part in the regulation of gene expression. Putative G4 forming sequences (PQS) have been reported in mammals, yeast, bacteria, and viruses. ...Here, we present PQS searches on the genomes of T. brucei, L. major, and P. falciparum. We found telomeric sequences and new PQS motifs. Biophysical experiments showed that EBR1, a 29 nucleotide long highly repeated PQS in T. brucei, forms a stable G4 structure. G4 ligands based on carbohydrate conjugated naphthalene diimides (carb-NDIs) that bind G4’s including hTel could bind EBR1 with selectivity versus dsDNA. These ligands showed important antiparasitic activity. IC50 values were in the nanomolar range against T. brucei with high selectivity against MRC-5 human cells. Confocal microscopy confirmed these ligands localize in the nucleus and kinetoplast of T. brucei suggesting they can reach their potential G4 targets. Cytotoxicity and zebrafish toxicity studies revealed sugar conjugation reduces intrinsic toxicity of NDIs.
Telomeres protect chromosome ends from being recognized as double-stranded breaks. Telomeric function is ensured by the shelterin complex in which TRF2 protein is an essential player. The G-rich ...strand of telomere DNA can fold into G-quadruplex (G4) structure. Small molecules stabilizing G4 structures, named G4 ligands, have been shown to alter telomeric functions in human cells. In this study, we show that a guanine-rich RNA sequence located in the 5'-UTR region of the TRF2 mRNA (hereafter 91TRF2G) is capable of forming a stable quadruplex that causes a 2.8-fold decrease in the translation of a reporter gene in human cells, as compared to a mutant 5'-UTR unable to fold into G4. We also demonstrate that several highly selective G4 ligands, the pyridine dicarboxamide derivative 360A and bisquinolinium compounds Phen-DC(3) and Phen-DC(6), are able to bind the 91TRF2G:RNA sequence and to modulate TRF2 protein translation in vitro. Since the naturally occurring 5'-UTR TRF2:RNA G4 element was used here, which is conserved in several vertebrate orthologs, the present data substantiate a potential translational mechanism mediated by a G4 RNA motif for the downregulation of TRF2 expression.
Current multiagent chemotherapy regimens have improved the cure rate in acute leukemia patients, but they are highly toxic and poorly efficient in relapsed patients. To improve the treatment ...approaches, new specific molecules are needed. The G‐quadruplexes (G4s), which are noncanonical nucleic acid structures found in specific guanine‐rich DNA or RNA, are involved in many cellular events, including control of gene expression. G4s are considered as targets for the development of anticancer agents. Heterocyclic molecules are well known to target and stabilize G4 structures. Thus, a new series of 2,9‐bis(substituted‐aminomethyl)phenyl‐1,10‐phenanthroline derivatives (1a–i) was designed, synthesized, and evaluated against five human myeloid leukemia cell lines (K562, KU812, MV4‐11, HL60, and U937). Their ability to stabilize various oncogene promoter G4 structures (c‐MYC, BCL‐2, and K‐RAS) as well as the telomeric G4 was also determined through the fluorescence resonance energy transfer melting assay and native mass spectrometry. In addition, the more bioactive ligands 1g–i were tested for telomerase activity in HuT78 and MV4‐11 protein extracts.
2,9‐Bis(substituted‐aminomethyl)phenyl‐1,10‐phenanthrolines 1a–i were designed, synthesized, and evaluated against five human myeloid leukemia cell lines. Their ability to stabilize the c‐MYC, BCL‐2, and K‐RAS oncogene promoter G4 structures was determined by fluorescence resonance energy transfer melting assay and native mass spectrometry. Telomerase activity in HuT78 and MV4‐11 protein extracts was investigated in the presence of the more bioactive ligands.
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