scholarly journals A Novel G-Quadruplex Binding Protein in Yeast—Slx9

Molecules ◽  
2019 ◽  
Vol 24 (9) ◽  
pp. 1774 ◽  
Author(s):  
Silvia Götz ◽  
Satyaprakash Pandey ◽  
Sabrina Bartsch ◽  
Stefan Juranek ◽  
Katrin Paeschke
Keyword(s):  
Binding Protein ◽  
Sequence Motifs ◽  
Dna And Rna ◽  
G4 Dna ◽  
G Quadruplex ◽  
High Thermodynamic Stability ◽  
Guanine Base ◽  
Regulatory Functions

G-quadruplex (G4) structures are highly stable four-stranded DNA and RNA secondary structures held together by non-canonical guanine base pairs. G4 sequence motifs are enriched at specific sites in eukaryotic genomes, suggesting regulatory functions of G4 structures during different biological processes. Considering the high thermodynamic stability of G4 structures, various proteins are necessary for G4 structure formation and unwinding. In a yeast one-hybrid screen, we identified Slx9 as a novel G4-binding protein. We confirmed that Slx9 binds to G4 DNA structures in vitro. Despite these findings, Slx9 binds only insignificantly to G-rich/G4 regions in Saccharomyces cerevisiae as demonstrated by genome-wide ChIP-seq analysis. However, Slx9 binding to G4s is significantly increased in the absence of Sgs1, a RecQ helicase that regulates G4 structures. Different genetic and molecular analyses allowed us to propose a model in which Slx9 recognizes and protects stabilized G4 structures in vivo.

scholarly journals G-quadruplex DNA drives genomic instability and represents a targetable molecular abnormality in ATRX-deficient malignant glioma

2018 ◽  
Author(s):  
Yuxiang Wang ◽  
Jie Yang ◽  
Wei Wu ◽  
Rachna Shah ◽  
Carla Danussi ◽  
...  
Keyword(s):  
Dna Damage ◽  
Malignant Glioma ◽  
Model Systems ◽  
Copy Number Alterations ◽  
Molecular Alteration ◽  
Quadruplex Dna ◽  
G4 Dna ◽  
G Quadruplex

AbstractMutational inactivation of ATRX (α-thalassemia mental retardation X-linked) represents a defining molecular alteration in large subsets of malignant glioma. Yet the pathogenic consequences of ATRX deficiency remain unclear, as do tractable mechanisms for its therapeutic targeting. Here we report that ATRX loss in isogenic glioma model systems induces replication stress and DNA damage by way of G-quadruplex (G4) DNA secondary structure. Moreover, these effects are associated with the acquisition of disease-relevant copy number alterations over time. We then demonstrate, both in vitro and in vivo, that ATRX deficiency selectively enhances DNA damage and cell death following chemical G4 stabilization. Finally, we show that G4 stabilization synergizes with other DNA-damaging therapies, including ionizing radiation, in the ATRX-deficient context. Our findings reveal novel pathogenic mechanisms driven by ATRX deficiency in glioma, while also pointing to tangible strategies for drug development.

scholarly journals The Functional Consequences of Eukaryotic Topoisomerase 1 Interaction with G-Quadruplex DNA

Genes ◽  
2020 ◽  
Vol 11 (2) ◽  
pp. 193
Author(s):  
Alexandra Berroyer ◽  
Nayun Kim
Keyword(s):  
Topoisomerase I ◽  
Genome Instability ◽  
Strand Break ◽  
Dna Topology ◽  
Single Strand Break ◽  
Dna Structures ◽  
G4 Dna ◽  
G Quadruplex

Topoisomerase I in eukaryotic cells is an important regulator of DNA topology. Its catalytic function is to remove positive or negative superhelical tension by binding to duplex DNA, creating a reversible single-strand break, and finally religating the broken strand. Proper maintenance of DNA topological homeostasis, in turn, is critically important in the regulation of replication, transcription, DNA repair, and other processes of DNA metabolism. One of the cellular processes regulated by the DNA topology and thus by Topoisomerase I is the formation of non-canonical DNA structures. Non-canonical or non-B DNA structures, including the four-stranded G-quadruplex or G4 DNA, are potentially pathological in that they interfere with replication or transcription, forming hotspots of genome instability. In this review, we first describe the role of Topoisomerase I in reducing the formation of non-canonical nucleic acid structures in the genome. We further discuss the interesting recent discovery that Top1 and Top1 mutants bind to G4 DNA structures in vivo and in vitro and speculate on the possible consequences of these interactions.

scholarly journals Mammalian CST averts replication failure by preventing G-quadruplex accumulation

2018 ◽  
Author(s):  
Yang Liu ◽  
Miaomiao Zhang ◽  
Bing Wang ◽  
Yingnan Xiao ◽  
Tingfang Li ◽  
...  
Keyword(s):  
Replication Fork ◽  
Genome Integrity ◽  
G4 Dna ◽  
Genome Wide ◽  
G Quadruplex ◽  
Preferential Loss ◽  
Telomere Replication ◽  
Lagging Strand

AbstractHuman CST (CTC1-STN1-TEN1) is an RPA-like complex that associates with G-rich single-strand DNA and helps resolve replication problems both at telomeres and genome-wide. We previously showed that CST binds and disrupts G-quadruplex (G4) DNA in vitro, suggesting that CST may prevent in vivo blocks to replication by resolving G4 structures. Here, we demonstrate that CST binds and unfolds G4 with similar efficiency to RPA. In cells, CST is recruited to telomeric and non-telomeric chromatin upon G4 stabilization. STN1 depletion increases G4 accumulation and slows bulk genomic DNA replication. At telomeres, combined STN1 depletion and G4 stabilization causes multi-telomere FISH signals and telomere loss, hallmarks of deficient telomere duplex replication. Strand-specific telomere FISH indicates preferential loss of C-strand DNA while analysis of BrdU uptake during leading and lagging-strand telomere replication shows preferential under-replication of lagging telomeres. Together these results indicate a block to Okazaki fragment synthesis. Overall, our findings indicate a novel role for CST in maintaining genome integrity through resolution of G4 structures both ahead of the replication fork and on the lagging strand template.

scholarly journals High Affinity binding of Yeast Nucleolin Nsr1 to Co-transcriptionally Formed G4 DNA Obstructs Replication and Elevates Genome Instability

2019 ◽  
Author(s):  
Shivani Singh ◽  
Alexandra Berroyer ◽  
Minseon Kim ◽  
Nayun Kim
Keyword(s):  
Dna Binding ◽  
Genome Instability ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
Yeast Genome ◽  
Stable Complex ◽  
High Affinity ◽  
G4 Dna ◽  
G Quadruplex

ABSTRACTA significant increase in genome instability is associated with the conformational shift of a guanine-run-containing DNA strand into the four-stranded G-quadruplex (G4) DNA. The mechanism underlying the recombination and genome rearrangements following the formation of G4 DNA in vivo has been difficult to elucidate but has become better clarified by the identification and functional characterization of several key G4 DNA-binding proteins. Mammalian nucleolin NCL is a highly specific G4 DNA-binding protein with a well-defined role in the transcriptional regulation of genes with associated G4 DNA-forming sequence motifs at their promoters. The consequence of the in vivo interaction between G4 DNA and nucleolin in respect to the genome instability has not been previously investigated. We show here that G4 DNA-binding is a conserved function in the yeast nucleolin Nsr1. Furthermore, we demonstrate that the Nsr1-G4 DNA complex formation results in replication obstruction and is a major factor in inducing the genome instability associated with the co-transcriptionally formed G4 DNA in the yeast genome. The G4-associated genome instability and the G4 DNA-binding in vivo requires the arginine-glycine-glycine (RGG) repeats located at the C-terminus of the Nsr1 protein. Nsr1 with the deletion of RGG domain supports normal cell growth and is sufficient for its pre-rRNA processing function. However, the truncation of RGG domain of Nsr1 significantly weakens its interaction with G4 DNA in vitro and in vivo and restores unhindered replication, overall resulting in a sharp reduction in the G4-associated genome instability. Our data suggest that the interaction between Nsr1 with the intact RGG repeats and G4 DNA impairs genome stability by precluding the access of G4-resolving proteins and obstructing replication.AUTHOR SUMMARYGenome instability is uniquely elevated at sequences containing multiple runs of guanines, which can fold into the unusual, four-stranded G-quadruplex (G4) DNA. In this study, we report a novel finding that a highly conserved G4 DNA binding protein Nsr1 can elevate the rate of recombination and chromosomal rearrangement occurring at a G4 DNA-forming sequence in the genome of Saccharomyces cerevisiae. The elevated genome instability requires the C-terminally located RGG domain of Nsr1, which supports the high-affinity interaction between the protein and G4 DNA. The connection between G4-specific genome instability and the function of Nsr1 to form stable complex with G4 DNA led to the hypothesis that the high-affinity Nsr1-G4 DNA complexes can become a barrier to replication. We demonstrate here that the presence of Nsr1 in fact slows the replication past a G4 DNA-containing genomic site and that the RGG domain is required to facilitate such replication block.

scholarly journals The Host Factor Polyhedrin Promoter Binding Protein (PPBP) Is Involved in Transcription from the Baculovirus Polyhedrin Gene Promoter

1998 ◽  
Vol 72 (9) ◽  
pp. 7484-7493 ◽  
Author(s):  
Sudip Ghosh ◽  
Anjali Jain ◽  
Bipasha Mukherjee ◽  
Saman Habib ◽  
Seyed E. Hasnain
Keyword(s):  
Binding Protein ◽  
Nuclear Extract ◽  
In Vitro Transcription ◽  
Host Factor ◽  
Luciferase Reporter ◽  
Sequence Motifs ◽  
Luciferase Reporter Gene ◽  
Polyhedrin Promoter

ABSTRACT Hypertranscription and temporal expression from theAutographa californica nuclear polyhedrosis (AcNPV) baculovirus polyhedrin promoter involves an α-amanitin-resistant RNA polymerase and requires a trans-acting viral factor(s). We previously reported that a 30-kDa host factor, polyhedrin promoter binding protein (PPBP), binds with unusual affinity, specificity, and stability to the transcriptionally important motif AATAAATAAGTATT within the polyhedrin (polh) initiator promoter and also displays coding strand-specific single-stranded DNA (ssDNA)-binding activity (S. Burma, B. Mukherjee, A. Jain, S. Habib, and S. E. Hasnain, J. Biol. Chem. 269:2750–2757, 1994; B. Mukherjee, S. Burma, and S. E. Hasnain, J. Biol. Chem. 270:4405–4411, 1995). We now present evidence which indicates that an additional factor(s) is involved in stabilizing PPBP-duplex promoter and PPBP-ssDNA interactions. TBP (TATA box binding protein) present in Spodoptera frugiperda(Sf9) cells is characteristically distinct from PPBP and does not interact directly with the polhpromoter. Replacement of PPBP cognate sequences within thepolh promoter with random nucleotides abolished PPBP binding in vitro and also failed to express the luciferase reporter gene in vivo. Phosphocellulose fractions of total nuclear extract from virus-infected cells which support in vitro transcription from thepolh promoter contain PPBP activity. When PPBP was sequestered by the presence of oligonucleotides containing PPBP cognate sequence motifs, in vitro transcription of a C-free reporter cassette was affected but was restored by the exogenous addition of nuclear extract containing PPBP. When PPBP was mopped out in vivo by a plasmid carrying PPBP cognate sequence present intrans, polh promoter-driven expression of the luciferase reporter was abolished, demonstrating that binding of PPBP to the polh promoter is essential for transcription.

scholarly journals E. coli Rep helicase and RecA recombinase unwind G4 DNA and are important for resistance to G4-stabilizing ligands

2020 ◽  
Vol 48 (12) ◽  
pp. 6640-6653 ◽  
Author(s):  
Tapas Paul ◽  
Andrew F Voter ◽  
Rachel R Cueny ◽  
Momčilo Gavrilov ◽  
Taekjip Ha ◽  
...  
Keyword(s):  
Atp Hydrolysis ◽  
Cellular Toxicity ◽  
Dna Structures ◽  
E Coli ◽  
G4 Dna ◽  
G Quadruplex ◽  
Physical Barriers ◽  
Stabilizing Ligands

Abstract G-quadruplex (G4) DNA structures can form physical barriers within the genome that must be unwound to ensure cellular genomic integrity. Here, we report unanticipated roles for the Escherichia coli Rep helicase and RecA recombinase in tolerating toxicity induced by G4-stabilizing ligands in vivo. We demonstrate that Rep and Rep-X (an enhanced version of Rep) display G4 unwinding activities in vitro that are significantly higher than the closely related UvrD helicase. G4 unwinding mediated by Rep involves repetitive cycles of G4 unfolding and refolding fueled by ATP hydrolysis. Rep-X and Rep also dislodge G4-stabilizing ligands, in agreement with our in vivo G4-ligand sensitivity result. We further demonstrate that RecA filaments disrupt G4 structures and remove G4 ligands in vitro, consistent with its role in countering cellular toxicity of G4-stabilizing ligands. Together, our study reveals novel genome caretaking functions for Rep and RecA in resolving deleterious G4 structures.

scholarly journals Telomere Function and the G-Quadruplex Formation are Regulated by hnRNP U

Cells ◽  
2019 ◽  
Vol 8 (5) ◽  
pp. 390 ◽  
Author(s):  
Hiroto Izumi ◽  
Keiko Funa
Keyword(s):  
Mammalian Cells ◽  
Binding Protein ◽  
Protein A ◽  
Western Blots ◽  
Telomere Repeat ◽  
G Quadruplex ◽  
Hnrnp U ◽  
Telomere Biology

We examine the role of the heterogenous ribonucleoprotein U (hnRNP U) as a G-quadruplex binding protein in human cell lines. Hypothesizing that hnRNP U is associated with telomeres, we investigate what other telomere-related functions it may have. Telomeric G-quadruplexes have been fully characterized in vitro, but until now no clear evidence of their function or in vivo interactions with proteins has been revealed in mammalian cells. Techniques used were immunoprecipitation, DNA pull-down, binding assay, and Western blots. We identified hnRNP U as a G-quadruplex binding protein. Immunoprecipitations disclosed that endogenous hnRNP U associates with telomeres, and DNA pull-downs showed that the hnRNP U C-terminus specifically binds telomeric G-quadruplexes. We have compared the effect of telomere repeat containing RNA (TERRA) on binding between hnRNP U and telomeric (Tel) or single- stranded Tel (ssTel) oligonucleotides and found that ssTel binds stronger to TERRA than to Tel. We also show that hnRNP U prevents replication protein A (RPA) accumulation at telomeres, and the recognition of telomeric ends by hnRNP suggests that a G-quadruplex promoting protein regulates its accessibility. Thus, hnRNP U-mediated formation has important functions for telomere biology.

scholarly journals Plasmodium falciparum GBP2 is a telomere-associated protein that binds to G-quadruplex DNA and RNA

2021 ◽  
Author(s):  
James Edwards-Smallbone ◽  
Anders L Jensen ◽  
Lydia E Roberts ◽  
Francis Isidore Garcia Totanes ◽  
Sarah R Hart ◽  
...  
Keyword(s):  
Rna Binding ◽  
Protozoan Parasite ◽  
Telomere Maintenance ◽  
Model Systems ◽  
Quadruplex Dna ◽  
Dna And Rna ◽  
G Quadruplex ◽  
Associated Proteins

In the early-diverging protozoan parasite Plasmodium, few telomere-binding proteins have been identified and several are unique. Plasmodium telomeres, like those of most eukaryotes, contain guanine-rich repeats that can form G-quadruplex structures. In model systems, quadruplex-binding drugs can disrupt telomere maintenance and some quadruplex-binding drugs are potent anti-plasmodial agents. Therefore, telomere-interacting and quadruplex-interacting proteins may offer new targets for anti-malarial therapy. Here, we report that P. falciparum GBP2 is such a protein. It was identified via Proteomics of Isolated Chromatin fragments, applied here for the first time in Plasmodium. In vitro, PfGBP2 binds specifically to G-rich telomere repeats in quadruplex form and it can also bind to G-rich RNA. In vivo, PfGBP2 partially colocalises with the known telomeric protein HP1 but is also found in the cytoplasm, probably due to its affinity for RNA. Consistently, its interactome includes numerous RNA-associated proteins. PfGBP2 is evidently a multifunctional DNA/RNA-binding factor in Plasmodium.

scholarly journals Identification of optimal fluorescent probes for G-quadruplex nucleic acids through systematic exploration of mono- and distyryl dye libraries

2019 ◽  
Vol 15 ◽  
pp. 1872-1889 ◽  
Author(s):  
Xiao Xie ◽  
Michela Zuffo ◽  
Marie-Paule Teulade-Fichou ◽  
Anton Granzhan
Keyword(s):  
Rna Structure ◽  
Stokes Shift ◽  
Topological Classification ◽  
Fluorescent Light ◽  
Styryl Dyes ◽  
Dna And Rna ◽  
Double Stranded Dna ◽  
G4 Dna ◽  
G Quadruplex

A library of 52 distyryl and 9 mono-styryl cationic dyes was synthesized and investigated with respect to their optical properties, propensity to aggregation in aqueous medium, and capacity to serve as fluorescence “light-up” probes for G-quadruplex (G4) DNA and RNA structures. Among the 61 compounds, 57 dyes showed preferential enhancement of fluorescence intensity in the presence of one or another G4-DNA or RNA structure, while no dye displayed preferential response to double-stranded DNA or single-stranded RNA analytes employed at equivalent nucleotide concentration. Thus, preferential fluorimetric response towards G4 structures appears to be a common feature of mono- and distyryl dyes, including long-known mono-styryl dyes used as mitochondrial probes or protein stains. However, the magnitude of the G4-induced “light-up” effect varies drastically, as a function of both the molecular structure of the dyes and the nature or topology of G4 analytes. Although our results do not allow to formulate comprehensive structure–properties relationships, we identified several structural motifs, such as indole- or pyrrole-substituted distyryl dyes, as well as simple mono-stryryl dyes such as DASPMI [2-(4-(dimethylamino)styryl)-1-methylpyridinium iodide] or its 4-isomer, as optimal fluorescent light-up probes characterized by high fluorimetric response (I/I 0 of up to 550-fold), excellent selectivity with respect to double-stranded DNA or single-stranded RNA controls, high quantum yield in the presence of G4 analytes (up to 0.32), large Stokes shift (up to 150 nm) and, in certain cases, structural selectivity with respect to one or another G4 folding topology. These dyes can be considered as promising G4-responsive sensors for in vitro or imaging applications. As a possible application, we implemented a simple two-dye fluorimetric assay allowing rapid topological classification of G4-DNA structures.

scholarly journals Seminal Plasma: Relevant for Fertility?

2021 ◽  
Vol 22 (9) ◽  
pp. 4368
Author(s):  
Heriberto Rodriguez-Martinez ◽  
Emilio A. Martinez ◽  
Juan J. Calvete ◽  
Fernando J. Peña Vega ◽  
Jordi Roca
Keyword(s):  
Seminal Plasma ◽  
Current Knowledge ◽  
Inorganic Ions ◽  
Vitro Fertilization ◽  
Dna And Rna ◽  
Model Animal ◽  
Sexual Glands ◽  
Species Specific

Seminal plasma (SP), the non-cellular component of semen, is a heterogeneous composite fluid built by secretions of the testis, the epididymis and the accessory sexual glands. Its composition, despite species-specific anatomical peculiarities, consistently contains inorganic ions, specific hormones, proteins and peptides, including cytokines and enzymes, cholesterol, DNA and RNA—the latter often protected within epididymis- or prostate-derived extracellular vesicles. It is beyond question that the SP participates in diverse aspects of sperm function pre-fertilization events. The SP also interacts with the various compartments of the tubular genital tract, triggering changes in gene function that prepares for an eventual successful pregnancy; thus, it ultimately modulates fertility. Despite these concepts, it is imperative to remember that SP-free spermatozoa (epididymal or washed ejaculated) are still fertile, so this review shall focus on the differences between the in vivo roles of the SP following semen deposition in the female and those regarding additions of SP on spermatozoa handled for artificial reproduction, including cryopreservation, from artificial insemination to in vitro fertilization. This review attempts, including our own results on model animal species, to critically summarize the current knowledge of the reproductive roles played by SP components, particularly in our own species, which is increasingly affected by infertility. The ultimate goal is to reconcile the delicate balance between the SP molecular concentration and their concerted effects after temporal exposure in vivo. We aim to appraise the functions of the SP components, their relevance as diagnostic biomarkers and their value as eventual additives to refine reproductive strategies, including biotechnologies, in livestock models and humans.

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