Characterization of G-quadruplex antibody reveals differential specificity for G4 DNA forms

Abstract Accumulating evidence suggests that human genome can fold into non-B DNA structures, when appropriate sequence and favourable conditions are present. Among these, G-quadruplexes (G4-DNA) are associated with gene regulation, chromosome fragility and telomere maintenance. Although several techniques are used in detecting such structures in vitro, understanding their intracellular existence has been challenging. Recently, an antibody, BG4, was described to study G4 structures within cells. Here, we characterize BG4 for its affinity towards G4-DNA, using several biochemical and biophysical tools. BG4 bound to G-rich DNA derived from multiple genes that form G-quadruplexes, unlike complementary C-rich or random sequences. BLI studies revealed robust binding affinity (Kd = 17.4 nM). Gel shift assays show BG4 binds to inter- and intramolecular G4-DNA, when it is in parallel orientation. Mere presence of G4-motif in duplex DNA is insufficient for antibody recognition. Importantly, BG4 can bind to G4-DNA within telomere sequence in a supercoiled plasmid. Finally, we show that BG4 binds to form efficient foci in four cell lines, irrespective of their lineage, demonstrating presence of G4-DNA in genome. Importantly, number of BG4 foci within the cells can be modulated, upon knockdown of G4-resolvase, WRN. Thus, we establish specificity of BG4 towards G4-DNA and discuss its potential applications.

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G-quadruplex DNA inhibits unwinding activity but promotes liquid–liquid phase separation by the DEAD-box helicase Ded1p

Chemical Communications ◽

10.1039/d1cc01479j ◽

2021 ◽

Author(s):

Jun Gao ◽

Zhaofeng Gao ◽

Andrea A. Putnam ◽

Alicia K. Byrd ◽

Sarah L. Venus ◽

...

Keyword(s):

Phase Separation ◽

Liquid Phase ◽

Liquid Phase Separation ◽

Intrinsically Disordered ◽

Dead Box ◽

G4 Dna ◽

G Quadruplex ◽

The Dead ◽

Liquid Liquid Phase Separation

G-quadruplex (G4) DNA inhibits RNA unwinding activity but promotes liquid–liquid phase separation of the DEAD-box helicase Ded1p in vitro and in cells. This highlights multifaceted effects of G4DNA on an enzyme with intrinsically disordered domains.

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G-quadruplex–R-loop interactions and the mechanism of anticancer G-quadruplex binders

Nucleic Acids Research ◽

10.1093/nar/gkaa944 ◽

2020 ◽

Vol 48 (21) ◽

pp. 11942-11957

Author(s):

Giulia Miglietta ◽

Marco Russo ◽

Giovanni Capranico

Keyword(s):

Selective Inhibition ◽

Telomere Maintenance ◽

Published Data ◽

Gene Promoters ◽

Untranslated Exon ◽

G Quadruplex ◽

Functional Consequences ◽

Hoogsteen Hydrogen Bonds

Abstract Genomic DNA and cellular RNAs can form a variety of non-B secondary structures, including G-quadruplex (G4) and R-loops. G4s are constituted by stacked guanine tetrads held together by Hoogsteen hydrogen bonds and can form at key regulatory sites of eukaryote genomes and transcripts, including gene promoters, untranslated exon regions and telomeres. R-loops are 3-stranded structures wherein the two strands of a DNA duplex are melted and one of them is annealed to an RNA. Specific G4 binders are intensively investigated to discover new effective anticancer drugs based on a common rationale, i.e.: the selective inhibition of oncogene expression or specific impairment of telomere maintenance. However, despite the high number of known G4 binders, such a selective molecular activity has not been fully established and several published data point to a different mode of action. We will review published data that address the close structural interplay between G4s and R-loops in vitro and in vivo, and how these interactions can have functional consequences in relation to G4 binder activity. We propose that R-loops can play a previously-underestimated role in G4 binder action, in relation to DNA damage induction, telomere maintenance, genome and epigenome instability and alterations of gene expression programs.

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

10.1101/347542 ◽

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.

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Helicases FANCJ, RTEL1 and BLM Act on Guanine Quadruplex DNA in Vivo

Genes ◽

10.3390/genes10110870 ◽

2019 ◽

Vol 10 (11) ◽

pp. 870 ◽

Cited By ~ 4

Author(s):

Peter Lansdorp ◽

Niek van Wietmarschen

Keyword(s):

Gene Expression ◽

Genome Stability ◽

Cell Function ◽

Historical Context ◽

Telomere Maintenance ◽

Dna Structures ◽

G4 Dna ◽

Guanine Quadruplex

Guanine quadruplex (G4) structures are among the most stable secondary DNA structures that can form in vitro, and evidence for their existence in vivo has been steadily accumulating. Originally described mainly for their deleterious effects on genome stability, more recent research has focused on (potential) functions of G4 structures in telomere maintenance, gene expression, and other cellular processes. The combined research on G4 structures has revealed that properly regulating G4 DNA structures in cells is important to prevent genome instability and disruption of normal cell function. In this short review we provide some background and historical context of our work resulting in the identification of FANCJ, RTEL1 and BLM as helicases that act on G4 structures in vivo. Taken together these studies highlight important roles of different G4 DNA structures and specific G4 helicases at selected genomic locations and telomeres in regulating gene expression and maintaining genome stability.

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

Genes ◽

10.3390/genes11020193 ◽

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.

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Overview of RNA G-quadruplex structures

Acta Biochimica Polonica ◽

10.18388/abp.2016_1335 ◽

2017 ◽

Vol 63 (4) ◽

Cited By ~ 18

Author(s):

Magdalena Małgowska

Keyword(s):

Three Dimensional ◽

Telomere Maintenance ◽

Structural Studies ◽

Biological Functions ◽

Cellular Processes ◽

Novel Drugs ◽

G Quadruplex ◽

Conformational Diversity

G-quadruplexes are non-canonical secondary structures which may be formed by guanine rich sequences, both in vitro and in living cells. The number of biological functions assigned to these structural motifs has grown rapidly since the discovery of their involvement in the telomere maintenance. Knowledge of the three-dimensional structures of G-quadruplexes plays an important role in understanding their conformational diversity, physiological functions, and in the design of novel drugs targeting G-quadruplexes. For the last decades, structural studies have been mainly focused on the DNA G-quadruplexes. Their RNA counterparts gained an increased interest along with still-emerging recognition of the central role of RNA in multiple cellular processes. In this review we focus on structural properties of RNA G-quadruplexes, based on high-resolution structures, available in RCSB PDB data base and on structural models. In addition, we point out to the current challenges in this field of research.

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Human Rev1 polymerase disrupts G-quadruplex DNA

Nucleic Acids Research ◽

10.1093/nar/gkt1314 ◽

2013 ◽

Vol 42 (5) ◽

pp. 3272-3285 ◽

Cited By ~ 37

Author(s):

Sarah Eddy ◽

Amit Ketkar ◽

Maroof K. Zafar ◽

Leena Maddukuri ◽

Jeong-Yun Choi ◽

...

Keyword(s):

Genome Maintenance ◽

Quadruplex Dna ◽

Dna Structures ◽

G4 Dna ◽

G Quadruplex ◽

Steady State Rate ◽

Dna Substrates ◽

Successful Replication ◽

Y Family

Abstract The Y-family DNA polymerase Rev1 is required for successful replication of G-quadruplex DNA (G4 DNA) in higher eukaryotes. Here we show that human Rev1 (hRev1) disrupts G4 DNA structures and prevents refolding in vitro. Nucleotidyl transfer by hRev1 is not necessary for mechanical unfolding to occur. hRev1 binds G4 DNA substrates with Kd,DNA values that are 4–15-fold lower than those of non-G4 DNA substrates. The pre-steady-state rate constant of deoxycytidine monophosphate (dCMP) insertion opposite the first tetrad-guanine by hRev1 is ∼56% as fast as that observed for non-G4 DNA substrates. Thus, hRev1 can promote fork progression by either dislodging tetrad guanines to unfold the G4 DNA, which could assist in extension by other DNA polymerases, or hRev1 can prevent refolding of G4 DNA structures. The hRev1 mechanism of action against G-quadruplexes helps explain why replication progress is impeded at G4 DNA sites in Rev1-deficient cells and illustrates another unique feature of this enzyme with important implications for genome maintenance.

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Mammalian CST averts replication failure by preventing G-quadruplex accumulation

10.1101/354050 ◽

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.

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The Relevance of G-Quadruplexes for DNA Repair

International Journal of Molecular Sciences ◽

10.3390/ijms222212599 ◽

2021 ◽

Vol 22 (22) ◽

pp. 12599

Author(s):

Rebecca Linke ◽

Michaela Limmer ◽

Stefan Juranek ◽

Annkristin Heine ◽

Katrin Paeschke

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Genome Stability ◽

Genome Instability ◽

Genetic Disorders ◽

Telomere Maintenance ◽

Dna Structures ◽

G4 Dna ◽

G Quadruplex ◽

Repair Pathways

DNA molecules can adopt a variety of alternative structures. Among these structures are G-quadruplex DNA structures (G4s), which support cellular function by affecting transcription, translation, and telomere maintenance. These structures can also induce genome instability by stalling replication, increasing DNA damage, and recombination events. G-quadruplex-driven genome instability is connected to tumorigenesis and other genetic disorders. In recent years, the connection between genome stability, DNA repair and G4 formation was further underlined by the identification of multiple DNA repair proteins and ligands which bind and stabilize said G4 structures to block specific DNA repair pathways. The relevance of G4s for different DNA repair pathways is complex and depends on the repair pathway itself. G4 structures can induce DNA damage and block efficient DNA repair, but they can also support the activity and function of certain repair pathways. In this review, we highlight the roles and consequences of G4 DNA structures for DNA repair initiation, processing, and the efficiency of various DNA repair pathways.

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Response of Sulfolobus solfataricus Dpo4 polymerase in vitro to a DNA G-quadruplex

Mutagenesis ◽

10.1093/mutage/gez010 ◽

2019 ◽

Vol 34 (3) ◽

pp. 289-297 ◽

Cited By ~ 1

Author(s):

Alexandra Berroyer ◽

Gloria Alvarado ◽

Erik D Larson

Keyword(s):

Repetitive Dna ◽

Dna Sequences ◽

Sulfolobus Solfataricus ◽

Repetitive Dna Sequences ◽

Polymerase Eta ◽

G4 Dna ◽

G Quadruplex ◽

Dna Polymerase Iv ◽

Y Family

Abstract Repetitive DNA sequences support the formation of structures that can interrupt replication and repair, leading to breaks and mutagenesis. One particularly stable structure is G-quadruplex (G4) DNA, which is four-stranded and formed from tandemly repetitive guanine bases. When folded within a template, G4 interferes with DNA synthesis. Similar to non-duplex structures, DNA base lesions can also halt an advancing replication fork, but the Y-family polymerases solve this problem by bypassing the damage. In order to better understand how guanine-rich DNA is replicated, we have investigated the activity of the model Y-family polymerase, Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4), on guanine-rich templates in vitro. We find that Dpo4 progression on templates containing either a single GC-rich hairpin or a G4 DNA structure is greatly reduced and synthesis stalls at the structure. Human polymerase eta (hPol eta) showed the same pattern of stalling at G4; however, and in contrast to Klenow, hPol eta and Dpo4 partially synthesise into the guanine repeat. Substitution of the nucleotide selectivity residue in Dpo4 with alanine permitted ribonucleotide incorporation on unstructured templates, but this further reduced the ability of Dpo4 to synthesise across from the guanine repeats. The advancement of Dpo4 on G4 templates was highest when the reaction was supplied with only deoxycytidine triphosphate, suggesting that high-fidelity synthesis is favoured over misincorporation. Our results are consistent with a model where the Y-family polymerases pause upon encountering G4 structures but have an ability to negotiate some synthesis through tetrad-associated guanines. This suggests that the Y-family polymerases reduce mutagenesis by catalysing the accurate replication of repetitive DNA sequences, but most likely in concert with additional replication and structure resolution activities.

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