scholarly journals Small-molecule G-quadruplex stabilizers reveal a novel pathway of autophagy regulation in neurons

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Jose F Moruno-Manchon ◽  
Pauline Lejault ◽  
Yaoxuan Wang ◽  
Brenna McCauley ◽  
Pedram Honarpisheh ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Memory Deficits ◽  
Dna Helicase ◽  
Vitro Assay ◽  
Dna Structures ◽  
First Intron ◽  
G4 Dna ◽  
G Quadruplex ◽  
Dna Stabilization

Guanine-rich DNA sequences can fold into four-stranded G-quadruplex (G4-DNA) structures. G4-DNA regulates replication and transcription, at least in cancer cells. Here, we demonstrate that, in neurons, pharmacologically stabilizing G4-DNA with G4 ligands strongly downregulates the Atg7 gene. Atg7 is a critical gene for the initiation of autophagy that exhibits decreased transcription with aging. Using an in vitro assay, we show that a putative G-quadruplex-forming sequence (PQFS) in the first intron of the Atg7 gene folds into a G4. An antibody specific to G4-DNA and the G4-DNA-binding protein PC4 bind to the Atg7 PQFS. Mice treated with a G4 stabilizer develop memory deficits. Brain samples from aged mice contain G4-DNA structures that are absent in brain samples from young mice. Overexpressing the G4-DNA helicase Pif1 in neurons exposed to the G4 stabilizer improves phenotypes associated with G4-DNA stabilization. Our findings indicate that G4-DNA is a novel pathway for regulating autophagy in neurons.

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 Human Rev1 polymerase disrupts G-quadruplex DNA

2013 ◽  
Vol 42 (5) ◽  
pp. 3272-3285 ◽  
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.

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

Mutagenesis ◽  
2019 ◽  
Vol 34 (3) ◽  
pp. 289-297 ◽  
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.

Human Pif1 helicase is a G-quadruplex DNA-binding protein with G-quadruplex DNA-unwinding activity

2010 ◽  
Vol 430 (1) ◽  
pp. 119-128 ◽  
Author(s):  
Cyril M. Sanders
Keyword(s):  
Dna Binding ◽  
Dna Sequences ◽  
Genome Stability ◽  
Dna Helicase ◽  
Dna Unwinding ◽  
Helicase Domain ◽  
Quadruplex Dna ◽  
G4 Dna ◽  
G Quadruplex ◽  
Pif1 Helicase

Pif1 proteins are helicases that in yeast are implicated in the maintenance of genome stability. One activity of Saccharomyces cerevisiae Pif1 is to stabilize DNA sequences that could otherwise form deleterious G4 (G-quadruplex) structures by acting as a G4 resolvase. The present study shows that human Pif1 (hPif1, nuclear form) is a G4 DNA-binding and resolvase protein and that these activities are properties of the conserved helicase domain (amino acids 206–620 of 641, hPifHD). hPif1 preferentially bound synthetic G4 DNA relative to ssDNA (single-stranded DNA), dsDNA (double-stranded DNA) and a partially single-stranded duplex DNA helicase substrate. G4 DNA unwinding, but not binding, required an extended (>10 nucleotide) 5′ ssDNA tail, and in competition assays, G4 DNA was an ineffective suppressor of helicase activity compared with ssDNA. These results suggest a distinction between the determinants of G4 DNA binding and the ssDNA interactions required for helicase action and that hPif1 may act on G4 substrates by binding alone or as a resolvase. Human Pif1 could therefore have a role in processing G4 structures that arise in the single-stranded nucleic acid intermediates formed during DNA replication and gene expression.

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 Mitochondrial genetic variation is enriched in G-quadruplex regions that stall DNA synthesis in vitro

2020 ◽  
Vol 29 (8) ◽  
pp. 1292-1309 ◽  
Author(s):  
Thomas J Butler ◽  
Katrina N Estep ◽  
Joshua A Sommers ◽  
Robert W Maul ◽  
Ann Zenobia Moore ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Genetic Variation ◽  
Dna Replication ◽  
Dna Synthesis ◽  
Dna Sequences ◽  
Computational Analysis ◽  
Italian Population ◽  
Dna Structures ◽  
G Quadruplex

Abstract As the powerhouses of the eukaryotic cell, mitochondria must maintain their genomes which encode proteins essential for energy production. Mitochondria are characterized by guanine-rich DNA sequences that spontaneously form unusual three-dimensional structures known as G-quadruplexes (G4). G4 structures can be problematic for the essential processes of DNA replication and transcription because they deter normal progression of the enzymatic-driven processes. In this study, we addressed the hypothesis that mitochondrial G4 is a source of mutagenesis leading to base-pair substitutions. Our computational analysis of 2757 individual genomes from two Italian population cohorts (SardiNIA and InCHIANTI) revealed a statistically significant enrichment of mitochondrial mutations within sequences corresponding to stable G4 DNA structures. Guided by the computational analysis results, we designed biochemical reconstitution experiments and demonstrated that DNA synthesis by two known mitochondrial DNA polymerases (Pol γ, PrimPol) in vitro was strongly blocked by representative stable G4 mitochondrial DNA structures, which could be overcome in a specific manner by the ATP-dependent G4-resolving helicase Pif1. However, error-prone DNA synthesis by PrimPol using the G4 template sequence persisted even in the presence of Pif1. Altogether, our results suggest that genetic variation is enriched in G-quadruplex regions that impede mitochondrial DNA replication.

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

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.

scholarly journals ATRX promotes heterochromatin formation to protect cells from G-quadruplex DNA-mediated stress

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Yu-Ching Teng ◽  
Aishwarya Sundaresan ◽  
Ryan O’Hara ◽  
Vincent U. Gant ◽  
Minhua Li ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Genome Stability ◽  
Helicase Domain ◽  
Quadruplex Dna ◽  
Heterochromatin Formation ◽  
Replicative Stress ◽  
G4 Dna ◽  
Mutation Burden ◽  
G Quadruplex ◽  
Dna Replication Stress

AbstractATRX is a tumor suppressor that has been associated with protection from DNA replication stress, purportedly through resolution of difficult-to-replicate G-quadruplex (G4) DNA structures. While several studies demonstrate that loss of ATRX sensitizes cells to chemical stabilizers of G4 structures, the molecular function of ATRX at G4 regions during replication remains unknown. Here, we demonstrate that ATRX associates with a number of the MCM replication complex subunits and that loss of ATRX leads to G4 structure accumulation at newly synthesized DNA. We show that both the helicase domain of ATRX and its H3.3 chaperone function are required to protect cells from G4-induced replicative stress. Furthermore, these activities are upstream of heterochromatin formation mediated by the histone methyltransferase, ESET, which is the critical molecular event that protects cells from G4-mediated stress. In support, tumors carrying mutations in either ATRX or ESET show increased mutation burden at G4-enriched DNA sequences. Overall, our study provides new insights into mechanisms by which ATRX promotes genome stability with important implications for understanding impacts of its loss on human disease.

scholarly journals Subtle structural alterations in G-quadruplex DNA regulate site specificity of fluorescence light-up probes

2020 ◽  
Vol 48 (3) ◽  
pp. 1108-1119 ◽  
Author(s):  
Rajendra Kumar ◽  
Karam Chand ◽  
Sudipta Bhowmik ◽  
Rabindra Nath Das ◽  
Snehasish Bhattacharjee ◽  
...  
Keyword(s):  
Rational Design ◽  
Dna Structure ◽  
Biological Processes ◽  
Chemical Research ◽  
Quadruplex Dna ◽  
Dna Structures ◽  
Future Studies ◽  
G4 Dna ◽  
G Quadruplex ◽  
Fluorescence Light

Abstract G-quadruplex (G4) DNA structures are linked to key biological processes and human diseases. Small molecules that target specific G4 DNA structures and signal their presence would therefore be of great value as chemical research tools with potential to further advance towards diagnostic and therapeutic developments. However, the development of these types of specific compounds remain as a great challenge. In here, we have developed a compound with ability to specifically signal a certain c-MYC G4 DNA structure through a fluorescence light-up mechanism. Despite the compound's two binding sites on the G4 DNA structure, only one of them result in the fluorescence light-up effect. This G-tetrad selectivity proved to originate from a difference in flexibility that affected the binding affinity and tilt the compound out of the planar conformation required for the fluorescence light-up mechanism. The intertwined relation between the presented factors is likely the reason for the lack of examples using rational design to develop compounds with turn-on emission that specifically target certain G4 DNA structures. However, this study shows that it is indeed possible to develop such compounds and present insights into the molecular details of specific G4 DNA recognition and signaling to advance future studies of G4 biology.

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.

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