Telomeric Overhang Length Determines Structural Dynamics and Accessibility to Telomerase and ALT-Associated Proteins

Hwang, Helen; Kreig, Alex; Calvert, Jacob; Lormand, Justin D; Kwon, Yong-Ho; Daley, James M.; Sung, Patrick; Opresko, Patricia L.; Myong, Sua

doi:10.1016/j.str.2014.03.013

Cited by 69 publications

(116 citation statements)

References 88 publications

(104 reference statements)

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“…Given these advantages, SiMPull may prove to be a valuable tool for plant researchers conducting interaction studies with endogenous proteins as well as those limited by antibodies with low affinities. In addition, unlike most assays, SiMPull is not limited solely to protein-protein interactions but can also quantitatively interrogate interactions between proteins and compounds, such as nucleic acids, small molecule ligands, and lipids (Jain et al, 2011(Jain et al, , 2014Krishnan et al, 2013;Hwang et al, 2014aHwang et al, , 2014bRodgers et al, 2015;Arauz et al, 2016).…”

Section: Discussionmentioning

confidence: 99%

In Planta Single-Molecule Pull-Down Reveals Tetrameric Stoichiometry of HD-ZIPIII:LITTLE ZIPPER Complexes

Husbands

Aggarwal

et al. 2016

The Plant Cell

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Deciphering complex biological processes markedly benefits from approaches that directly assess the underlying biomolecular interactions. Most commonly used approaches to monitor protein-protein interactions typically provide nonquantitative readouts that lack statistical power and do not yield information on the heterogeneity or stoichiometry of protein complexes. Single-molecule pull-down (SiMPull) uses single-molecule fluorescence detection to mitigate these disadvantages and can quantitatively interrogate interactions between proteins and other compounds, such as nucleic acids, small molecule ligands, and lipids. Here, we establish SiMPull in plants using the HOMEODOMAIN LEUCINE ZIPPER III (HD-ZIPIII) and LITTLE ZIPPER (ZPR) interaction as proof-of-principle. Colocalization analysis of fluorophore-tagged HD-ZIPIII and ZPR proteins provides strong statistical evidence of complex formation. In addition, we use SiMPull to directly quantify YFP and mCherry maturation probabilities, showing these differ substantially from values obtained in mammalian systems. Leveraging these probabilities, in conjunction with fluorophore photobleaching assays on over 2000 individual complexes, we determined HD-ZIPIII:ZPR stoichiometry. Intriguingly, these complexes appear as heterotetramers, comprising two HD-ZIPIII and two ZPR molecules, rather than heterodimers as described in the current model. This surprising result raises new questions about the regulation of these key developmental factors and is illustrative of the unique contribution SiMPull is poised to make to in planta protein interaction studies.

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Section: Discussionmentioning

confidence: 99%

In Planta Single-Molecule Pull-Down Reveals Tetrameric Stoichiometry of HD-ZIPIII:LITTLE ZIPPER Complexes

Husbands

Aggarwal

et al. 2016

The Plant Cell

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“…H69 and H93 tandem structures clearly display complex melting profiles (the first derivative of their absorbance with respect to temperature has a peak at 61 C and a shoulder around 40 C), suggesting that they may be composed of G4 units of different stabilities. When representing the G4 structures formed by (GGGTTA) 3,7,11,15 GGG sequences as the sum of G4 units suitably flanked by TTA sequences (Fig. 3A), it is notable that they are not equivalent in terms of G4 units composing them.…”

Section: Formation and Stability Of Contiguous G4s By (Gggtta) 7111mentioning

confidence: 99%

“…Likely a TTA tail, depending on its position (5 0 side, 3 0 side or both sides), drives the folding of the whole sequence into different G4 conformations characterized by different stabilities, as suggested by nuclear magnetic resonance (NMR) studies on TA-H21, H21-T(T) and (T)TA-H21-TT sequences [17,19e22,32]. TTA(GGGTTA) 15 The impact of TTA tails on the stability of an individual G4 suggests that, in the G4 tandem structures formed by (GGGTTA) 4mÀ1 GGG sequences (m ¼ 2, 3, 4, …), the 5 0 terminal G4 (mimicked by H21-TTA) has a higher stability than the 3 0 terminal G4 (mimicked by TTA-H21), while the inner G4 units (mimicked by TTA-H21-TTA) have the lowest stability (Fig. 3A).…”

Section: Formation and Stability Of Contiguous G4s By (Gggtta) 7111mentioning

confidence: 99%

“…In mammalian cells, indirect evidence suggests that G4s may occur at telomeres during replication and at the 3 0 -overhangs, challenging telomere maintenance, at least in the absence of proteins able to unfold G4s or in the presence of ligands able to stabilize these structures [7e10]. Telomeric G4s modulate in vitro telomerase activity [11e13] and binding of other proteins involved in telomere biology, as recently demonstrated by single-molecule approaches [14,15].…”

Section: Introductionmentioning

confidence: 99%

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Understanding the stability of DNA G-quadruplex units in long human telomeric strands

Bugaut

Alberti

2015

Biochimie

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Human telomeric DNA is composed of GGGTTA repeats. The presence of consecutive guanines makes the telomeric G-strand prone to fold into contiguous (or tandem) G-quadruplexes (G4s). The aim of this study was to provide a clarified picture of the stability of telomeric tandem G4 structures as a function of the number of G4 units and of boundary sequences, and an understanding of the diversity of their melting behaviors in terms of the single G4 units composing them. To this purpose we undertook an UV-spectroscopic investigation of the structure and stability of telomeric repeats potentially able to fold into up to four contiguous G4s, flanked or not by TTA sequences at their 5' and 3' extremities. We explain why the stability of (GGGTTA)4m-1GGG structures (m = 2, 3, 4 …) decreases with increasing the number m of G4 units, whereas the stability of TTA-(GGGTTA)4m-1GGG-TTA structures does not. Our results support that the inner G4 units have similar stabilities, whereas the stabilities of the terminal G4 units are modulated by their flanking nucleotides: in a TTA-(GGGTTA)4m-1GGG-TTA tandem context, the terminal G4 units are roughly as stable as the inner G4 units; while in a (GGGTTA)4m-1GGG tandem context, the G4 at the 5' extremity is more stable than the G4 at the 3' extremity, which in turn is more stable than an inner G4. Our study provides new information about the global and local stability of telomeric tandem G4 structures under near physiological conditions.

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“…It rescues cancer cells from end-replication problem by maintaining telomere length using its intrinsic RNA as the template [19], thus conferring a strong selective advantage for continued growth of malignant cells [20][21][22]. The guanine-rich, single strand terminal ends of telomeres may fold into a strong and stable intramolecular G4 structures, which prevents accessibility to telomerase [23]. Various strategies have targeted this structure to suppress telomerase activity in rapidly dividing cancer cells [24][25][26].…”

Section: Introductionmentioning

confidence: 99%

Selectivity of major isoquinoline alkaloids from Chelidonium majus towards telomeric G-quadruplex: A study using a transition-FRET (t-FRET) assay

Noureini

Esmaeili

Abachi

et al. 2017

Biochimica et Biophysica Acta (BBA) - General Subjects

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BackgroundNatural bioproducts are invaluable resources in drug discovery. Isoquinoline alkaloids of Chelidonium majus constitute a structurally diverse family of natural products that are of great interest, one of them being their selectivity for human telomeric G-quadruplex structure and telomerase inhibition. MethodsThe study focuses on the mechanism of telomerase inhibition by stabilization of telomeric Gquadruplex structures by berberine, chelerythrine, chelidonine, sanguinarine and papaverine. Telomerase activity and mRNA levels of hTERT were estimated using quantitative telomere repeat amplification protocol and qPCR, in MCF-7 cells treated with different groups of alkaloids. The selectivity of the main isoquinoline alkaloids of Chelidonium majus towards telomeric Gquadruplex forming sequences were explored using a sensitive modified thermal FRET-melting measurement in the presence of the complementary oligonucleotide CT22. We assessed and monitored G-quadruplex topologies using circular dichroism (CD) methods, and compared spectra to previously well-characterized motifs, either alone or in the presence of the alkaloids, Molecular modeling was performed to rationalize ligand binding to the G-quadruplex structure. ResultsThe results highlight strong inhibitory effects of chelerythrine, sanguinarine and berberine on telomerase activity, most likely through substrate sequestration. These isoquinoline alkaloids interacted strongly with telomeric sequence G-quadruplex. In comparison, chelidonine and papaverine had no significant interaction with the telomeric quadruplex, while they strongly inhibited telomerase at transcription level of hTERT. Altogether, all of the studied alkaloids showed various levels and mechanisms of telomerase inhibition. ConclusionsWe report on a comparative study of anti-telomerase activity of the isoquinoline alkaloids of Chelidonium majus. Chelerythrine was most effective in inhibiting telomerase activity by substrate sequesteration through G-quadruplex stabilization. General SignificanceUnderstanding structural and molecular mechanisms of anti-cancer agents can help in developing new and more potent drugs with fewer side effects. Isoquinolines are the most biologically active agents from Chelidonium majus, which have shown to be telomeric G-quadruplex stabilizers and potent telomerase inhibitors.

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Telomeric Overhang Length Determines Structural Dynamics and Accessibility to Telomerase and ALT-Associated Proteins

Cited by 69 publications

References 88 publications

In Planta Single-Molecule Pull-Down Reveals Tetrameric Stoichiometry of HD-ZIPIII:LITTLE ZIPPER Complexes

In Planta Single-Molecule Pull-Down Reveals Tetrameric Stoichiometry of HD-ZIPIII:LITTLE ZIPPER Complexes

Understanding the stability of DNA G-quadruplex units in long human telomeric strands

Selectivity of major isoquinoline alkaloids from Chelidonium majus towards telomeric G-quadruplex: A study using a transition-FRET (t-FRET) assay

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