The Yeast Pif1 Helicase Prevents Genomic Instability Caused by G-Quadruplex-Forming CEB1 Sequences In Vivo

G-rich sequences found at multiple sites throughout all genomes may form secondary structures called G-quadruplexes (G4), which act as roadblocks for molecular motors. Among the enzymes thought to process these structures, the Pif1 DNA helicase is considered as an archetypical G4-resolvase and its absence has been linked to G4-related genomic instabilities in yeast. Here we developed a single-molecule assay to observe Pif1 opening a DNA duplex and resolving the G4 in real time. In support of former enzymological studies, we show that the helicase reduces the lifetime of G4 from hours to seconds. However, we observe that in presence of a G4, Pif1 exhibits a strong strand switching behavior, which can lead to Pif1 escaping G4 resolution, depending on the structural context surrounding the substrate. This behavior is also detected in presence of other roadblocks (LNA or RNA). We propose that the efficiency of Pif1 to remove a roadblock (G4 or other) is affected by its strand switching behavior and depends on the context surrounding the obstacle. We discuss how this switching behavior may explain several aspects of Pif1 substrate preference and affect its activity as a G4 resolvase in vivo.

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Astrocytes derived from p53-deficient mice provide a multistep in vitro model for development of malignant gliomas.

Molecular and Cellular Biology ◽

10.1128/mcb.15.8.4249 ◽

1995 ◽

Vol 15 (8) ◽

pp. 4249-4259 ◽

Cited By ~ 65

Author(s):

A M Yahanda ◽

J M Bruner ◽

L A Donehower ◽

R S Morrison

Keyword(s):

Genomic Instability ◽

Malignant Transformation ◽

Nude Mice ◽

Malignant Gliomas ◽

Early Event ◽

Wild Type ◽

Early Passage ◽

Wild Type P53

Loss or mutation of p53 is thought to be an early event in the malignant transformation of many human astrocytic tumors. To better understand the role of p53 in their growth and transformation, we developed a model employing cultured neonatal astrocytes derived from mice deficient in one (p53 +/-) or both (p53 -/-) p53 alleles, comparing them with wild-type (p53 +/+) cells. Studies of in vitro and in vivo growth and transformation were performed, and flow cytometry and karyotyping were used to correlate changes in growth with genomic instability. Early-passage (EP) p53 -/- astrocytes achieved higher saturation densities and had more rapid growth than EP p53 +/- and +/+ cells. The EP p53 -/- cells were not transformed, as they were unable to grow in serum-free medium or in nude mice. With continued passaging, p53 -/- cells exhibited a multistep progression to a transformed phenotype. Late-passage p53 -/- cells achieved saturation densities 50 times higher than those of p53 +/+ cells and formed large, well-vascularized tumors in nude mice. p53 +/- astrocytes exhibited early loss of the remaining wild-type p53 allele and then evolved in a manner phenotypically similar to p53 -/- astrocytes. In marked contrast, astrocytes retaining both wild-type p53 alleles never exhibited a transformed phenotype and usually senesced after 7 to 10 passages. Dramatic alterations in ploidy and karyotype occurred and were restricted to cells deficient in wild-type p53 following repeated passaging. The results of these studies suggest that loss of wild-type p53 function promotes genomic instability, accelerated growth, and malignant transformation in astrocytes.

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Up-Regulating Relaxin Expression by G-Quadruplex Interactive Ligand to Achieve Antifibrotic Action

Endocrinology ◽

10.1210/en.2012-1114 ◽

2012 ◽

Vol 153 (8) ◽

pp. 3692-3700 ◽

Cited By ~ 28

Author(s):

Hui-Ping Gu ◽

Sen Lin ◽

Ming Xu ◽

Hai-Yi Yu ◽

Xiao-Jun Du ◽

...

Keyword(s):

Rna Polymerase Ii ◽

Cardiac Fibrosis ◽

Cardiac Fibroblasts ◽

Heart Diseases ◽

Gene Promoter ◽

Binding Motif ◽

Endogenous Expression ◽

G Quadruplex

Myocardial fibrosis is a key pathological change in a variety of heart diseases contributing to the development of heart failure, arrhythmias, and sudden death. Recent studies have shown that relaxin prevents and reverses cardiac fibrosis. Endogenous expression of relaxin was elevated in the setting of heart disease; the extent of such up-regulation, however, is insufficient to exert compensatory actions, and the mechanism regulating relaxin expression is poorly defined. In the rat relaxin-1 (RLN1, Chr1) gene promoter region we found presence of repeated guanine (G)-rich sequences, which allowed formation and stabilization of G-quadruplexes with the addition of a G-quadruplex interactive ligand berberine. The G-rich sequences and the G-quadruplexes were localized adjacent to the binding motif of signal transducer and activator of transcription (STAT)3, which negatively regulates relaxin expression. Thus, we hypothesized that the formation and stabilization of G-quadruplexes by berberine could influence relaxin expression. We found that berberine-induced formation of G-quadruplexes did increase relaxin gene expression measured at mRNA and protein levels. Formation of G-quadruplexes significantly reduced STAT3 binding to the promoter of relaxin gene. This was associated with consequent increase in the binding of RNA polymerase II and STAT5a to relaxin gene promoter. In cardiac fibroblasts and rats treated with angiotensin II, berberine was found to suppress fibroblast activation, collagen synthesis, and extent of cardiac fibrosis through up-regulating relaxin. The antifibrotic action of berberine in vitro and in vivo was similar to that by exogenous relaxin. Our findings document a novel therapeutic strategy for fibrosis through up-regulating expression of endogenous relaxin.

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Erratum: Corrigendum: Structural revisions of small molecules reported to cross-link G-quadruplex DNA in vivo reveal a repetitive assignment error in the literature

Scientific Reports ◽

10.1038/srep40471 ◽

2017 ◽

Vol 7 (1) ◽

Author(s):

Paul E. Reyes-Gutiérrez ◽

Tomáš Kapal ◽

Blanka Klepetářová ◽

David Šaman ◽

Radek Pohl ◽

...

Keyword(s):

Small Molecules ◽

Cross Link ◽

Quadruplex Dna ◽

G Quadruplex ◽

Assignment Error

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V(D)J-mediated Translocations in Lymphoid Neoplasms: A Functional Assessment of Genomic Instability by Cryptic Sites⋆

Journal of Experimental Medicine ◽

10.1084/jem.20011578 ◽

2002 ◽

Vol 195 (1) ◽

pp. 85-98 ◽

Cited By ~ 109

Author(s):

Rodrig Marculescu ◽

Trang Le ◽

Paul Simon ◽

Ulrich Jaeger ◽

Bertrand Nadel

Keyword(s):

T Cell ◽

Genomic Instability ◽

Lymphoblastic Leukemia ◽

Cell Receptor ◽

Lymphoid Malignancies ◽

Cell Acute Lymphoblastic Leukemia ◽

Normal Human ◽

Hodgkin's Lymphomas ◽

The Impact

Most lymphoid malignancies are initiated by specific chromosomal translocations between immunoglobulin (Ig)/T cell receptor (TCR) gene segments and cellular proto-oncogenes. In many cases, illegitimate V(D)J recombination has been proposed to be involved in the translocation process, but this has never been functionally established. Using extra-chromosomal recombination assays, we determined the ability of several proto-oncogenes to target V(D)J recombination, and assessed the impact of their recombinogenic potential on translocation rates in vivo. Our data support the involvement of 2 distinct mechanisms: translocations involving LMO2, TAL2, and TAL1 in T cell acute lymphoblastic leukemia (T-ALL), are compatible with illegitimate V(D)J recombination between a TCR locus and a proto-oncogene locus bearing a fortuitous but functional recombination site (type 1); in contrast, translocations involving BCL1 and BCL2 in B cell non-Hodgkin’s lymphomas (B-NHL), are compatible with a process in which only the IgH locus breaks are mediated by V(D)J recombination (type 2). Most importantly, we show that the t(11;14)(p13;q32) translocation involving LMO2 is present at strikingly high frequency in normal human thymus, and that the recombinogenic potential conferred by the LMO2 cryptic site is directly predictive of the in vivo level of translocation at that locus. These findings provide new insights into the regulation forces acting upon genomic instability in B and T cell tumorigenesis.

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Porphyrins to restrict progression of pancreatic cancer by stabilizing KRAS G-quadruplex: In silico, in vitro and in vivo validation of anticancer strategy

European Journal of Pharmaceutical Sciences ◽

10.1016/j.ejps.2018.09.011 ◽

2018 ◽

Vol 125 ◽

pp. 39-53 ◽

Cited By ~ 9

Author(s):

Rudradip Pattanayak ◽

Atish Barua ◽

Amlan Das ◽

Tanima Chatterjee ◽

Adrija Pathak ◽

...

Keyword(s):

Pancreatic Cancer ◽

In Silico ◽

G Quadruplex ◽

In Vivo Validation

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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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4. AAV Labeling of Break-Prone Palindromes in Mammalian Genomes: Exploitation of the In Vivo rAAV Biology for Studies of Genomic Instability in Living Animals

Molecular Therapy ◽

10.1016/s1525-0016(16)44210-3 ◽

2007 ◽

Vol 15 ◽

pp. S2

Keyword(s):

Genomic Instability ◽

Mammalian Genomes

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On the interaction of an anticancer trisubstituted naphthalene diimide with G-quadruplexes of different topologies: a structural insight

Nucleic Acids Research ◽

10.1093/nar/gkaa1001 ◽

2020 ◽

Vol 48 (21) ◽

pp. 12380-12393

Author(s):

Chiara Platella ◽

Marko Trajkovski ◽

Filippo Doria ◽

Mauro Freccero ◽

Janez Plavec ◽

...

Keyword(s):

Anticancer Activity ◽

Dissociation Constants ◽

Therapeutic Potential ◽

In Vivo Studies ◽

Binding Modes ◽

Hybrid Topology ◽

Naphthalene Diimide ◽

G Quadruplex ◽

Naphthalene Diimides

Abstract Naphthalene diimides showed significant anticancer activity in animal models, with therapeutic potential related to their ability to strongly interact with G-quadruplexes. Recently, a trifunctionalized naphthalene diimide, named NDI-5, was identified as the best analogue of a mini-library of novel naphthalene diimides for its high G-quadruplex binding affinity along with marked, selective anticancer activity, emerging as promising candidate drug for in vivo studies. Here we used NMR, dynamic light scattering, circular dichroism and fluorescence analyses to investigate the interactions of NDI-5 with G-quadruplexes featuring either parallel or hybrid topology. Interplay of different binding modes of NDI-5 to G-quadruplexes was observed for both parallel and hybrid topologies, with end-stacking always operative as the predominant binding event. While NDI-5 primarily targets the 5′-end quartet of the hybrid G-quadruplex model (m-tel24), the binding to a parallel G-quadruplex model (M2) occurs seemingly simultaneously at the 5′- and 3′-end quartets. With parallel G-quadruplex M2, NDI-5 formed stable complexes with 1:3 DNA:ligand binding stoichiometry. Conversely, when interacting with hybrid G-quadruplex m-tel24, NDI-5 showed multiple binding poses on a single G-quadruplex unit and/or formed different complexes comprising two or more G-quadruplex units. NDI-5 produced stabilizing effects on both G-quadruplexes, forming complexes with dissociation constants in the nM range.

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