scholarly journals Emerging roles for R-loop structures in the management of topological stress

2020 ◽  
Vol 295 (14) ◽  
pp. 4684-4695 ◽  
Author(s):  
Frederic Chedin ◽  
Craig J. Benham
Keyword(s):  
Dna Sequence ◽  
Single Molecule ◽  
Genome Instability ◽  
Dna Topology ◽  
Loop Structure ◽  
Loop Modeling ◽  
Loop Formation ◽  
Dna Structures ◽  
Recent Developments ◽  
Dna Template

R-loop structures are a prevalent class of alternative non-B DNA structures that form during transcription upon invasion of the DNA template by the nascent RNA. R-loops form universally in the genomes of organisms ranging from bacteriophages, bacteria, and yeasts to plants and animals, including mammals. A growing body of work has linked these structures to both physiological and pathological processes, in particular to genome instability. The rising interest in R-loops is placing new emphasis on understanding the fundamental physicochemical forces driving their formation and stability. Pioneering work in Escherichia coli revealed that DNA topology, in particular negative DNA superhelicity, plays a key role in driving R-loops. A clear role for DNA sequence was later uncovered. Here, we review and synthesize available evidence on the roles of DNA sequence and DNA topology in controlling R-loop formation and stability. Factoring in recent developments in R-loop modeling and single-molecule profiling, we propose a coherent model accounting for the interplay between DNA sequence and DNA topology in driving R-loop structure formation. This model reveals R-loops in a new light as powerful and reversible topological stress relievers, an insight that significantly expands the repertoire of R-loops' potential biological roles under both normal and aberrant conditions.

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 Single-molecule fluorescence studies on cotranscriptional G-quadruplex formation coupled with R-loop formation

2020 ◽  
Vol 48 (16) ◽  
pp. 9195-9203
Author(s):  
Gunhyoung Lim ◽  
Sungchul Hohng
Keyword(s):  
Gene Regulation ◽  
Single Molecule ◽  
Genome Instability ◽  
Feedback Mechanism ◽  
Rnase H ◽  
Formation Mechanisms ◽  
Loop Formation ◽  
Single Molecule Fluorescence ◽  
Fluorescence Studies ◽  
G Quadruplex

Abstract G-quadruplex (GQ) is formed at various regions of DNA, including telomeres of chromosomes and regulatory regions of oncogenes. Since GQ is important in both gene regulation and genome instability, the biological and medical implications of this abnormal DNA structure have been intensively studied. Its formation mechanisms, however, are not clearly understood yet. We report single-molecule fluorescence experiments to monitor the cotranscriptional GQ formation coupled with R-loop formation using T7 RNA polymerase. The GQ is formed very rarely per single-round transcription. R-loop formation precedes and facilitates GQ formation. Once formed, some GQs are extremely stable, resistant even to RNase H treatment, and accumulate in multiple-round transcription conditions. On the other hand, GQ existing in the non-template strand promotes the R-loop formation in the next rounds of transcription. Our study clearly shows the existence of a positive feedback mechanism of GQ and R-loop formations, which may possibly contribute to gene regulation and genome instability.

scholarly journals RAD51AP1 mediates RAD51 activity through nucleosome interaction

2020 ◽  
Author(s):  
Elena Pires ◽  
Neelam Sharma ◽  
Platon Selemenakis ◽  
Bo Wu ◽  
Yuxin Huang ◽  
...  
Keyword(s):  
Genome Instability ◽  
Loop Formation ◽  
Synaptic Complex ◽  
Nucleosome Core ◽  
Histone Octamer ◽  
Nucleosome Core Particles ◽  
Dna Capture ◽  
Dna Template ◽  
Template Dna

AbstractRAD51 Associated Protein 1 (RAD51AP1) is a key protein in the homologous recombination DNA repair pathway (HR). Loss of RAD51AP1 leads to defective HR, genome instability and telomere erosion. RAD51AP1 physically interacts with the RAD51 recombinase and promotes RAD51-mediated capture of the donor DNA, synaptic complex assembly and displacement-loop formation when tested with synthetic, nucleosome-free DNA substratesin vitro. In cells, however, DNA is packaged into chromatin, posing an additional barrier to the complexities of the HR reaction. How RAD51AP1 functions as an HR activator in the context of chromatin has remained unclear.In this study, we show that RAD51AP1 binds to Nucleosome Core Particles (NCPs). We identified a C-terminal region in RAD51AP1 and its previously mapped DNA binding domain as critical for mediating the association between RAD51AP1 and both the NCP and the histone octamer. We show that RAD51AP1 is capable of promoting duplex DNA capture and initiating joint-molecule formation with the NCP and chromatinized template DNA, respectively. Together, our results suggest that RAD51AP1directlyassists the RAD51-mediated search of donor DNA in chromatin. We present a model, in which RAD51AP1 anchors the DNA template through affinity for its nucleosomes to the RAD51-ssDNA nucleoprotein filament.

scholarly journals TIN2 is an architectural protein that facilitates TRF2-mediated trans- and cis-interactions on telomeric DNA

2021 ◽  
Author(s):  
Parminder Kaur ◽  
Ryan Barnes ◽  
Hai Pan ◽  
Ariana C Detwiler ◽  
Ming Liu ◽  
...  
Keyword(s):  
Single Molecule ◽  
Molecular Model ◽  
Loop Formation ◽  
Dna Breaks ◽  
Telomeric Dna ◽  
Dna Structures ◽  
Shelterin Complex ◽  
Double Strand Dna Breaks ◽  
Architectural Protein

Abstract The telomere specific shelterin complex, which includes TRF1, TRF2, RAP1, TIN2, TPP1 and POT1, prevents spurious recognition of telomeres as double-strand DNA breaks and regulates telomerase and DNA repair activities at telomeres. TIN2 is a key component of the shelterin complex that directly interacts with TRF1, TRF2 and TPP1. In vivo, the large majority of TRF1 and TRF2 are in complex with TIN2 but without TPP1 and POT1. Since knockdown of TIN2 also removes TRF1 and TRF2 from telomeres, previous cell-based assays only provide information on downstream effects after the loss of TRF1/TRF2 and TIN2. Here, we investigated DNA structures promoted by TRF2–TIN2 using single-molecule imaging platforms, including tracking of compaction of long mouse telomeric DNA using fluorescence imaging, atomic force microscopy (AFM) imaging of protein–DNA structures, and monitoring of DNA–DNA and DNA–RNA bridging using the DNA tightrope assay. These techniques enabled us to uncover previously unknown unique activities of TIN2. TIN2S and TIN2L isoforms facilitate TRF2-mediated telomeric DNA compaction (cis-interactions), dsDNA–dsDNA, dsDNA–ssDNA and dsDNA–ssRNA bridging (trans-interactions). Furthermore, TIN2 facilitates TRF2-mediated T-loop formation. We propose a molecular model in which TIN2 functions as an architectural protein to promote TRF2-mediated trans and cis higher-order nucleic acid structures at telomeres.

scholarly journals Interplay between DNA sequence and negative superhelicity drives R-loop structures

2019 ◽  
Vol 116 (13) ◽  
pp. 6260-6269 ◽  
Author(s):  
Robert Stolz ◽  
Shaheen Sulthana ◽  
Stella R. Hartono ◽  
Maika Malig ◽  
Craig J. Benham ◽  
...  
Keyword(s):  
Dna Sequence ◽  
Single Molecule ◽  
Energy State ◽  
In Vitro Transcription ◽  
Loop Formation ◽  
Base Pairing ◽  
Theoretical Predictions ◽  
Nucleic Acid Structures ◽  
The Impact

R-loops are abundant three-stranded nucleic-acid structures that formin cisduring transcription. Experimental evidence suggests that R-loop formation is affected by DNA sequence and topology. However, the exact manner by which these factors interact to determine R-loop susceptibility is unclear. To investigate this, we developed a statistical mechanical equilibrium model of R-loop formation in superhelical DNA. In this model, the energy involved in forming an R-loop includes four terms—junctional and base-pairing energies and energies associated with superhelicity and with the torsional winding of the displaced DNA single strand around the RNA:DNA hybrid. This model shows that the significant energy barrier imposed by the formation of junctions can be overcome in two ways. First, base-pairing energy can favor RNA:DNA over DNA:DNA duplexes in favorable sequences. Second, R-loops, by absorbing negative superhelicity, partially or fully relax the rest of the DNA domain, thereby returning it to a lower energy state. In vitro transcription assays confirmed that R-loops cause plasmid relaxation and that negative superhelicity is required for R-loops to form, even in a favorable region. Single-molecule R-loop footprinting following in vitro transcription showed a strong agreement between theoretical predictions and experimental mapping of stable R-loop positions and further revealed the impact of DNA topology on the R-loop distribution landscape. Our results clarify the interplay between base sequence and DNA superhelicity in controlling R-loop stability. They also reveal R-loops as powerful and reversible topology sinks that cells may use to nonenzymatically relieve superhelical stress during transcription.

Curaxin-Induced DNA Topology Alterations Trigger the Distinct Binding Response of CTCF and FACT at the Single-Molecule Level

Biochemistry ◽  
2021 ◽  
Vol 60 (7) ◽  
pp. 494-499
Author(s):  
Ke Lu ◽  
Cuifang Liu ◽  
Yinuo Liu ◽  
Anfeng Luo ◽  
Jun Chen ◽  
...  
Keyword(s):  
Single Molecule ◽  
Dna Topology ◽  
Single Molecule Level

scholarly journals A machine learning approach for accurate and real-time DNA sequence identification

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Yiren Wang ◽  
Mashari Alangari ◽  
Joshua Hihath ◽  
Arindam K. Das ◽  
M. P. Anantram
Keyword(s):  
Real Time ◽  
Dna Sequence ◽  
Single Molecule ◽  
Identification Accuracy ◽  
Measuring System ◽  
Identification System ◽  
Genetic Sequencing ◽  
Sequence Identification ◽  
Tree Classifier ◽  
Single Mismatch

Abstract Background The all-electronic Single Molecule Break Junction (SMBJ) method is an emerging alternative to traditional polymerase chain reaction (PCR) techniques for genetic sequencing and identification. Existing work indicates that the current spectra recorded from SMBJ experimentations contain unique signatures to identify known sequences from a dataset. However, the spectra are typically extremely noisy due to the stochastic and complex interactions between the substrate, sample, environment, and the measuring system, necessitating hundreds or thousands of experimentations to obtain reliable and accurate results. Results This article presents a DNA sequence identification system based on the current spectra of ten short strand sequences, including a pair that differs by a single mismatch. By employing a gradient boosted tree classifier model trained on conductance histograms, we demonstrate that extremely high accuracy, ranging from approximately 96 % for molecules differing by a single mismatch to 99.5 % otherwise, is possible. Further, such accuracy metrics are achievable in near real-time with just twenty or thirty SMBJ measurements instead of hundreds or thousands. We also demonstrate that a tandem classifier architecture, where the first stage is a multiclass classifier and the second stage is a binary classifier, can be employed to boost the single mismatched pair’s identification accuracy to 99.5 %. Conclusions A monolithic classifier, or more generally, a multistage classifier with model specific parameters that depend on experimental current spectra can be used to successfully identify DNA strands.

scholarly journals Targeting Genome Stability in Melanoma—A New Approach to an Old Field

2021 ◽  
Vol 22 (7) ◽  
pp. 3485
Author(s):  
Marta Osrodek ◽  
Michal Wozniak
Keyword(s):  
Genome Stability ◽  
Genome Instability ◽  
Checkpoint Inhibitors ◽  
Mapk Pathway ◽  
Rapid Development ◽  
Melanoma Cells ◽  
Old Field ◽  
Number Of Patients ◽  
Recent Developments ◽  
Mapk Inhibitors

Despite recent groundbreaking advances in the treatment of cutaneous melanoma, it remains one of the most treatment-resistant malignancies. Due to resistance to conventional chemotherapy, the therapeutic focus has shifted away from aiming at melanoma genome stability in favor of molecularly targeted therapies. Inhibitors of the RAS/RAF/MEK/ERK (MAPK) pathway significantly slow disease progression. However, long-term clinical benefit is rare due to rapid development of drug resistance. In contrast, immune checkpoint inhibitors provide exceptionally durable responses, but only in a limited number of patients. It has been increasingly recognized that melanoma cells rely on efficient DNA repair for survival upon drug treatment, and that genome instability increases the efficacy of both MAPK inhibitors and immunotherapy. In this review, we discuss recent developments in the field of melanoma research which indicate that targeting genome stability of melanoma cells may serve as a powerful strategy to maximize the efficacy of currently available therapeutics.

scholarly journals Non-B DNA-Forming Motifs Promote Mfd-Dependent Stationary-Phase Mutagenesis in Bacillus subtilis

2021 ◽  
Vol 9 (6) ◽  
pp. 1284
Author(s):  
Tatiana Ermi ◽  
Carmen Vallin ◽  
Ana Gabriela Regalado García ◽  
Moises Bravo ◽  
Ismaray Fernandez Cordero ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Stationary Phase ◽  
Genome Instability ◽  
Mutagenic Effect ◽  
Genetic Changes ◽  
Dna Structures ◽  
Coding Regions ◽  
G4 Dna ◽  
Stressed Cells ◽  
Growing Conditions

Transcription-induced mutagenic mechanisms limit genetic changes to times when expression happens and to coding DNA. It has been hypothesized that intrinsic sequences that have the potential to form alternate DNA structures, such as non-B DNA structures, influence these mechanisms. Non-B DNA structures are promoted by transcription and induce genome instability in eukaryotic cells, but their impact in bacterial genomes is less known. Here, we investigated if G4 DNA- and hairpin-forming motifs influence stationary-phase mutagenesis in Bacillus subtilis. We developed a system to measure the influence of non-B DNA on B. subtilis stationary-phase mutagenesis by deleting the wild-type argF at its chromosomal position and introducing IPTG-inducible argF alleles differing in their ability to form hairpin and G4 DNA structures into an ectopic locus. Using this system, we found that sequences predicted to form non-B DNA structures promoted mutagenesis in B. subtilis stationary-phase cells; such a response did not occur in growing conditions. We also found that the transcription-coupled repair factor Mfd promoted mutagenesis at these predicted structures. In summary, we showed that non-B DNA-forming motifs promote genetic instability, particularly in coding regions in stressed cells; therefore, non-B DNA structures may have a spatial and temporal mutagenic effect in bacteria. This study provides insights into mechanisms that prevent or promote mutagenesis and advances our understanding of processes underlying bacterial evolution.

scholarly journals Rapid and multiplex preparation of engineered Mycobacterium smegmatis porin A (MspA) nanopores for single molecule sensing and sequencing

2021 ◽  
Author(s):  
Shuanghong Yan ◽  
Liying Wang ◽  
Xiaoyu Du ◽  
Shanyu Zhang ◽  
Sha Wang ◽  
...  
Keyword(s):  
Single Molecule ◽  
Mycobacterium Smegmatis ◽  
Recent Developments

Acknowledging its unique conical lumen structure, Mycobacterium smegmatis porin A (MspA) was the first type of nanopore that has successfully sequenced DNA. Recent developments of nanopore single molecule chemistry have...

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