scholarly journals Dynamic interaction of BRCA2 with telomeric G-quadruplexes underlies telomere replication homeostasis

2021 ◽  
Author(s):  
Junyeop Lee ◽  
Keewon Sung ◽  
So Young Joo ◽  
Jun-Hyeon Jeong ◽  
Seong Keun Kim ◽  
...  
Keyword(s):  
Single Molecule ◽  
Specific Interaction ◽  
Mobility Shift ◽  
Single Molecule Fret ◽  
C Terminus ◽  
G Quadruplex ◽  
Telomere Replication ◽  
Mobility Shift Assay ◽  
Stalled Replication Forks ◽  
Lagging Strand

Abstract BRCA2-deficient cells undergo telomere shortening upon collapse of stalled replication forks, particularly during lagging-strand telomere synthesis. The molecular mechanism underlying fork collapse remains unclear. Here we find that the BRCA2 C-terminus, which includes an OB-fold, specifically interacts with G-quadruplex (G4) structures generated during lagging-strand telomere replication. We demonstrate that BRCA2 associates with G-triplex (G3)-derived intermediates using electrophoretic mobility shift assay and single-molecule FRET. These G3 intermediates form during direct interconversion between parallel and non-parallel G4 structures. Intriguingly, MRE11 nuclease can resect G4-forming telomere sequences, a function that is inhibited by BRCA2. BRCA2 depletion consistently resulted in increased telomeric damage, which was relieved by MRE11 knockdown. These data suggest that BRCA2 interaction with telomeric G4 prevents MRE11-mediated resection. The specific interaction between BRCA2 and G4 therefore contributes to telomere stability and genome integrity.

scholarly journals RNA recognition and self-association of CPEB4 is mediated by its tandem RRM domains

2014 ◽  
Vol 42 (15) ◽  
pp. 10185-10195 ◽  
Author(s):  
Constanze Schelhorn ◽  
James M.B. Gordon ◽  
Lidia Ruiz ◽  
Javier Alguacil ◽  
Enrique Pedroso ◽  
...  
Keyword(s):  
Rna Binding ◽  
Titration Calorimetry ◽  
Rna Recognition Motif ◽  
Rna Recognition ◽  
Mobility Shift ◽  
Cytoplasmic Polyadenylation ◽  
C Terminus ◽  
Self Association ◽  
Mobility Shift Assay ◽  
A Minor

Abstract Cytoplasmic polyadenylation is regulated by the interaction of the cytoplasmic polyadenylation element binding proteins (CPEB) with cytoplasmic polyadenylation element (CPE) containing mRNAs. The CPEB family comprises four paralogs, CPEB1–4, each composed of a variable N-terminal region, two RNA recognition motif (RRM) and a C-terminal ZZ-domain. We have characterized the RRM domains of CPEB4 and their binding properties using a combination of biochemical, biophysical and NMR techniques. Isothermal titration calorimetry, NMR and electrophoretic mobility shift assay experiments demonstrate that both the RRM domains are required for an optimal CPE interaction and the presence of either one or two adenosines in the two most commonly used consensus CPE motifs has little effect on the affinity of the interaction. Both the single RRM1 and the tandem RRM1–RRM2 have the ability to dimerize, although representing a minor population. Self-association does not affect the proteins’ ability to interact with RNA as demonstrated by ion mobility–mass spectrometry. Chemical shift effects measured by NMR of the apo forms of the RRM1–RRM2 samples indicate that the two domains are orientated toward each other. NMR titration experiments show that residues on the β-sheet surface on RRM1 and at the C-terminus of RRM2 are affected upon RNA binding. We propose a model of the CPEB4 RRM1–RRM2–CPE complex that illustrates the experimental data.

scholarly journals Nucleolin stabilizes G-quadruplex structures folded by the LTR promoter and silences HIV-1 viral transcription

2015 ◽  
Vol 43 (18) ◽  
pp. 8884-8897 ◽  
Author(s):  
Elena Tosoni ◽  
Ilaria Frasson ◽  
Matteo Scalabrin ◽  
Rosalba Perrone ◽  
Elena Butovskaya ◽  
...  
Keyword(s):  
Transcriptional Activity ◽  
Cellular Protein ◽  
Spectrometric Analysis ◽  
Viral Transcription ◽  
Mobility Shift ◽  
Binding Preference ◽  
G Quadruplex ◽  
Mobility Shift Assay ◽  
Ltr Promoter ◽  
Hiv 1

Abstract Folding of the LTR promoter into dynamic G-quadruplex conformations has been shown to suppress its transcriptional activity in HIV-1. Here we sought to identify the proteins that control the folding of this region of proviral genome by inducing/stabilizing G-quadruplex structures. The implementation of electrophorethic mobility shift assay and pull-down experiments coupled with mass spectrometric analysis revealed that the cellular protein nucleolin is able to specifically recognize G-quadruplex structures present in the LTR promoter. Nucleolin recognized with high affinity and specificity the majority, but not all the possible G-quadruplexes folded by this sequence. In addition, it displayed greater binding preference towards DNA than RNA G-quadruplexes, thus indicating two levels of selectivity based on the sequence and nature of the target. The interaction translated into stabilization of the LTR G-quadruplexes and increased promoter silencing activity; in contrast, disruption of nucleolin binding in cells by both siRNAs and a nucleolin binding aptamer greatly increased LTR promoter activity. These data indicate that nucleolin possesses a specific and regulated activity toward the HIV-1 LTR promoter, which is mediated by G-quadruplexes. These observations provide new essential insights into viral transcription and a possible low mutagenic target for antiretroviral therapy.

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 Probing the kinetic and thermodynamic consequences of the tetraloop/tetraloop receptor monovalent ion-binding site in P4–P6 RNA by smFRET

2015 ◽  
Vol 43 (2) ◽  
pp. 172-178 ◽  
Author(s):  
Namita Bisaria ◽  
Daniel Herschlag
Keyword(s):  
Single Molecule ◽  
Specific Binding ◽  
Specific Interaction ◽  
Monovalent Cations ◽  
Folding Kinetics ◽  
Rna Molecules ◽  
Single Molecule Fret ◽  
Wide Range ◽  
Monovalent Ion

Structured RNA molecules play roles in central biological processes and understanding the basic forces and features that govern RNA folding kinetics and thermodynamics can help elucidate principles that underlie biological function. Here we investigate one such feature, the specific interaction of monovalent cations with a structured RNA, the P4–P6 domain of the Tetrahymena ribozyme. We employ single molecule FRET (smFRET) approaches as these allow determination of folding equilibrium and rate constants over a wide range of stabilities and thus allow direct comparisons without the need for extrapolation. These experiments provide additional evidence for specific binding of monovalent cations, Na+ and K+, to the RNA tetraloop–tetraloop receptor (TL–TLR) tertiary motif. These ions facilitate both folding and unfolding, consistent with an ability to help order the TLR for binding and further stabilize the tertiary contact subsequent to attainment of the folding transition state.

scholarly journals A comprehensive evaluation of a typical plant telomeric G-quadruplex (G4) DNA reveals the dynamics of G4 formation, rearrangement, and unfolding

2020 ◽  
Vol 295 (16) ◽  
pp. 5461-5469 ◽  
Author(s):  
Wen-Qiang Wu ◽  
Ming-Li Zhang ◽  
Chun-Peng Song
Keyword(s):  
Single Molecule ◽  
Comprehensive Evaluation ◽  
Cd Spectroscopy ◽  
Telomeric Sequence ◽  
Single Molecule Fret ◽  
G4 Dna ◽  
G Quadruplex ◽  
Simple Repeats ◽  
Plant Telomeres ◽  
Shed Light

Telomeres are specific nucleoprotein structures that are located at the ends of linear eukaryotic chromosomes and play crucial roles in genomic stability. Telomere DNA consists of simple repeats of a short G-rich sequence: TTAGGG in mammals and TTTAGGG in most plants. In recent years, the mammalian telomeric G-rich repeats have been shown to form G-quadruplex (G4) structures, which are crucial for modulating telomere functions. Surprisingly, even though plant telomeres are essential for plant growth, development, and environmental adaptions, only few reports exist on plant telomeric G4 DNA (pTG4). Here, using bulk and single-molecule assays, including CD spectroscopy, and single-molecule FRET approaches, we comprehensively characterized the structure and dynamics of a typical plant telomeric sequence, d[GGG(TTTAGGG)3]. We found that this sequence can fold into mixed G4s in potassium, including parallel and antiparallel structures. We also directly detected intermediate dynamic transitions, including G-hairpin, parallel G-triplex, and antiparallel G-triplex structures. Moreover, we observed that pTG4 is unfolded by the AtRecQ2 helicase but not by AtRecQ3. The results of our work shed light on our understanding about the existence, topological structures, stability, intermediates, unwinding, and functions of pTG4.

scholarly journals Targeting G-quadruplex Forming Sequences with Cas9

2020 ◽  
Author(s):  
Hamza Balci ◽  
Viktorija Globyte ◽  
Chirlmin Joo
Keyword(s):  
Single Molecule ◽  
Dna Sequences ◽  
Structural Characteristics ◽  
Structural Heterogeneity ◽  
Loop Formation ◽  
Conformational States ◽  
Single Molecule Fret ◽  
G Quadruplex ◽  
The Stability ◽  
The Impact

ABSTRACTClustered Regularly Interspaced Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) proteins, particularly Cas9, have provided unprecedented control on targeting and editing specific DNA sequences. If the target sequences are prone to folding into non-canonical secondary structures, such as G-quadruplex (GQ), the conformational states and activity of CRISPR-Cas9 complex would be influenced, but the impact has not been assessed. Using single molecule FRET, we investigated structural characteristics of the complex formed by CRISPR-Cas9 and target DNA, which contains a potentially GQ forming sequence (PQS) in either the target or the non-target strand (TS or NTS). We observed different conformational states and dynamics depending on the stability of the GQ and the position of PQS. When PQS was in NTS, we observed evidence for GQ formation for both weak and stable GQs. This is consistent with R-loop formation between TS and crRNA releasing NTS from Watson-Crick pairing and facilitating secondary structure formation in it. When PQS was in TS, R-loop formation was adequate to maintain a weak GQ in the unfolded state but not a GQ with moderate or high stability. The observed structural heterogeneity within the target dsDNA and the R-loop strongly depended on whether the PQS was in TS or NTS. We propose these variations in the complex structures to have functional implications for Cas9 activity.

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