scholarly journals BLM helicase facilitates telomere replication during leading strand synthesis of telomeres

2015 ◽  
Vol 210 (2) ◽  
pp. 191-208 ◽  
Author(s):  
William C. Drosopoulos ◽  
Settapong T. Kosiyatrakul ◽  
Carl L. Schildkraut
Keyword(s):  
Single Molecule ◽  
Werner Syndrome ◽  
Telomeric Dna ◽  
G4 Dna ◽  
Genome Wide ◽  
Blm Helicase ◽  
Telomere Replication ◽  
Leading Strand ◽  
Single Molecule Analysis

Based on its in vitro unwinding activity on G-quadruplex (G4) DNA, the Bloom syndrome–associated helicase BLM is proposed to participate in telomere replication by aiding fork progression through G-rich telomeric DNA. Single molecule analysis of replicated DNA (SMARD) was used to determine the contribution of BLM helicase to telomere replication. In BLM-deficient cells, replication forks initiating from origins within the telomere, which copy the G-rich strand by leading strand synthesis, moved slower through the telomere compared with the adjacent subtelomere. Fork progression through the telomere was further slowed in the presence of a G4 stabilizer. Using a G4-specific antibody, we found that deficiency of BLM, or another G4-unwinding helicase, the Werner syndrome-associated helicase WRN, resulted in increased G4 structures in cells. Importantly, deficiency of either helicase led to greater increases in G4 DNA detected in the telomere compared with G4 seen genome-wide. Collectively, our findings are consistent with BLM helicase facilitating telomere replication by resolving G4 structures formed during copying of the G-rich strand by leading strand synthesis.

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 Mapping DNA replication with nanopore sequencing

2018 ◽  
Author(s):  
Magali Hennion ◽  
Jean-Michel Arbona ◽  
Corinne Cruaud ◽  
Florence Proux ◽  
Benoît Le Tallec ◽  
...  
Keyword(s):  
Dna Replication ◽  
Single Molecule ◽  
Electrical Signal ◽  
Yeast Cells ◽  
Nanopore Sequencing ◽  
Single Molecule Level ◽  
Experimental Systems ◽  
Genome Wide ◽  
Single Molecule Analysis

ABSTRACTWe have harnessed nanopore sequencing to study DNA replication genome-wide at the single-molecule level. Using in vitro prepared DNA substrates, we characterized the effect of bromodeoxyuridine (BrdU) substitution for thymidine on the MinION nanopore electrical signal. Using a neural-network basecaller trained on yeast DNA containing either BrdU or thymidine, we identified BrdU-labelled tracts in yeast cells synchronously entering S phase in the presence of hydroxyurea and BrdU. As expected, the BrdU-labelled tracts coincided with previously identified early-firing, but not late-firing, replication origins. These results open the way to high-throughput, high-resolution, single-molecule analysis of DNA replication in many experimental systems.

scholarly journals Build Your Own Microscope: Step-By-Step Guide for Building a Prism-Based TIRF Microscope

2018 ◽  
Vol 1 (4) ◽  
pp. 40 ◽  
Author(s):  
Dalton Gibbs ◽  
Anisa Kaur ◽  
Anoja Megalathan ◽  
Kumar Sapkota ◽  
Soma Dhakal
Keyword(s):  
Single Molecule ◽  
Total Internal Reflection ◽  
Life Sciences ◽  
Inverted Microscope ◽  
Sophisticated Instrument ◽  
High Level ◽  
Single Molecule Analysis ◽  
Molecule Dynamics ◽  
Vast Range

Prism-based total internal reflection fluorescence (pTIRF) microscopy is one of the most widely used techniques for the single molecule analysis of a vast range of samples including biomolecules, nanostructures, and cells, to name a few. It allows for excitation of surface bound molecules/particles/quantum dots via evanescent field of a confined region of space, which is beneficial not only for single molecule detection but also for analysis of single molecule dynamics and for acquiring kinetics data. However, there is neither a commercial microscope available for purchase nor a detailed guide dedicated for building this microscope. Thus far, pTIRF microscopes are custom-built with the use of a commercially available inverted microscope, which requires high level of expertise in selecting and handling sophisticated instrument-parts. To directly address this technology gap, here we describe a step-by-step guide on how to build and characterize a pTIRF microscope for in vitro single-molecule imaging, nanostructure analysis and other life sciences research.

scholarly journals Beginning at the end: DNA replication within the telomere

2015 ◽  
Vol 210 (2) ◽  
pp. 177-179 ◽  
Author(s):  
Susan A. Gerbi
Keyword(s):  
Dna Replication ◽  
Mouse Chromosome ◽  
Single Molecule ◽  
Template Strand ◽  
Cell Biol ◽  
Chromosome Arm ◽  
Leading Strand ◽  
Wrn Helicase ◽  
Single Molecule Analysis

Using single molecule analysis of replicated DNA (SMARD), Drosopoulos et al. (2015; J. Cell Biol. http://dx.doi.org/10.1083/jcb.201410061) report that DNA replication initiates at measurable frequency within the telomere of mouse chromosome arm 14q. They demonstrate that resolution of G4 structures on the G-rich template strand of the telomere requires some overlapping functions of BLM and WRN helicase for leading strand synthesis.

scholarly journals Single-molecule analysis reveals self assembly and nanoscale segregation of two distinct cavin subcomplexes on caveolae

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Yann Gambin ◽  
Nicholas Ariotti ◽  
Kerrie-Ann McMahon ◽  
Michele Bastiani ◽  
Emma Sierecki ◽  
...  
Keyword(s):  
Single Molecule ◽  
Self Assembly ◽  
Mammalian Cells ◽  
Protein Complexes ◽  
Building Blocks ◽  
Scaffolding Protein ◽  
Cell Extracts ◽  
Single Molecule Analysis ◽  
Exquisite Specificity

In mammalian cells three closely related cavin proteins cooperate with the scaffolding protein caveolin to form membrane invaginations known as caveolae. Here we have developed a novel single-molecule fluorescence approach to directly observe interactions and stoichiometries in protein complexes from cell extracts and from in vitro synthesized components. We show that up to 50 cavins associate on a caveola. However, rather than forming a single coat complex containing the three cavin family members, single-molecule analysis reveals an exquisite specificity of interactions between cavin1, cavin2 and cavin3. Changes in membrane tension can flatten the caveolae, causing the release of the cavin coat and its disassembly into separate cavin1-cavin2 and cavin1-cavin3 subcomplexes. Each of these subcomplexes contain 9 ± 2 cavin molecules and appear to be the building blocks of the caveolar coat. High resolution immunoelectron microscopy suggests a remarkable nanoscale organization of these separate subcomplexes, forming individual striations on the surface of caveolae.

scholarly journals An Algorithmic framework for genome-wide identification of Sugarcane (Saccharum officinarum L.)-encoded microRNA targets against SCBV

2020 ◽  
Author(s):  
Muhammad Aleem Ashraf ◽  
Xiaoyan Feng ◽  
Xiaowen Hu ◽  
Fakiha Ashraf ◽  
Linbo Shen ◽  
...  
Keyword(s):  
Single Molecule ◽  
Hypothetical Protein ◽  
Saccharum Officinarum ◽  
Potential Candidate ◽  
Candidate Mirnas ◽  
Microrna Targets ◽  
Genome Wide ◽  
Algorithmic Framework ◽  
Sugarcane Bacilliform Virus

AbstractSugarcane Bacilliform Virus (SCBV) is considered an economically the most damaging pathogen for sugarcane production worldwide. Three ORFs are characterized in a single molecule of circular, ds-DNA genome of the SCBV, encoding for hypothetical protein (ORF1), DNA binding protein (ORF2) and Polyprotein (ORF3). The study was aimed to predict and comprehensively evaluate sugarcane miRNAs for the silencing of SCBV genome using in-silico algorithms. Computational methods were used for prediction of candidate miRNAs from sugarcane (S. officinarum L.) to silence the expression of SCBV genes through translational inhibition by mRNA cleavage. Mature sugarcane miRNAs were retrieved and were assessed to hybridization with the SCBV genome. A total of fourteen potential candidate miRNAs from sugarcane were computed by all the algorithms used for the silencing of SCBV. A consensus of three algorithms predicts hybridization sites of sof-miR159e at common locus 5534. The miRNA-mRNA interaction was estimated by computing free-energy of miRNA-mRNA duplex using RNAcofold algorithm. Regulatory network of predicted candidate miRNAs of sugarcane with SCBV ORFs, generated using Circos, identify novel targets. Consequently, detecting and discarding inefficient amiRNAs prior to cloning would help suppressed mutants faster. The efficacy of predicted candidate miRNAs was evaluated to test the survival rate of the in vitro amiRNA-mediated effective badnaviral silencing and resistance in sugarcane cultivars.

scholarly journals Folding and Persistence Time of Intramolecular G-Quadruplexes Transiently Embedded in a DNA duplex

2021 ◽  
Author(s):  
Phong Lan Thao Tran ◽  
Martin Rieu ◽  
Samar Hodeib ◽  
Alexandra Joubert ◽  
Jimmy Ouellet ◽  
...  
Keyword(s):  
Single Molecule ◽  
Critical Role ◽  
Genetic Regulation ◽  
Regulatory Elements ◽  
Promoter Regions ◽  
Folding Rate ◽  
Persistence Time ◽  
G4 Dna

ABSTRACTG-quadruplex (G4) DNA structures have emerged as important regulatory elements during DNA replication, transcription or repair. While many in-vitro studies have focused on the kinetics of G4 formation within DNA single-strands, G4 are found in-vivo in double-stranded DNA regions, where their formation is challenged by pairing between the two complementary strands. Since the energy of hybridization of Watson-Crick structures dominates the energy of G4 folding, this competition should play a critical role on the persistence of G4 in vivo. To address this issue, we designed a single molecule assay allowing measuring G4 folding and persistence while the structure is periodically challenged by the complementary strand. We quantified both the folding rate and the persistence time of biologically relevant G4 structures and showed that the dynamics of G4 formation depends strongly on the genomic location. G4 are found much more stable in promoter regions and replication origins than in telomeric regions. In addition, we characterized how G4 dynamics was affected by G4 ligands and showed that both folding rate and persistence increased. Our assay opens new perspectives for the measurement of G4 dynamics, which is critical to understand their role in genetic regulation.

Abstract 20439: Novel Long Intergenic Noncoding RNAs Modulate Adipose Functions in Human

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Xuan Zhang ◽  
Rachel Ballantyne ◽  
Chenyi Xue ◽  
Jane F Ferguson ◽  
Brian Gregory ◽  
...  
Keyword(s):  
Single Molecule ◽  
Noncoding Rnas ◽  
Cellular Functions ◽  
Protein Coding ◽  
Processing Bodies ◽  
P Bodies ◽  
Genome Wide ◽  
Potential Binding ◽  
Competing Endogenous Rnas

Recently long intergenic noncoding RNAs (lincRNAs) have emerged as key mediators of cellular differentiation and functions in a variety of cell systems critical to cardiovascular and metabolic disorders. To identify and investigate novel functional lincRNAs in human adipose, we performed deep high-throughput RNAseq (>200 million reads/sample) in subcutaneous adiposes of 13 health volunteers. Of an integrated dataset of 54,944 human lincRNAs, 6,558 lincRNAs were detected. Here we report 2 cytoplasmic adipose lincRNAs, linc-DMRT2 and linc-NFE2L3-1, were detected in human adipocytes but not monocytes or macrophages. Linc-DMRT2, one of the most abundant adipose lincRNAs, was markedly induced during in vitro human adipocyte differentiation. Notably, single molecule RNA FISH (fluorescence in situ hybridization) demonstrated that linc-DMRT2 were exclusively present in adipocyte cytoplasma and co-localized with processing bodies (P-bodies) marker, GW182, suggesting its potential role in modulating turnover of certain RNA species. In addition, linc-NFE2L3-1, predominantly detected in adipose and skeleton muscle, is localized near an established GWAS locus associated with waist-hip ratio adjusted BMI. We identified 4 SNPs in linc-NFE2L3-1 reaching genome wide significance for BMI (lead SNP rs10267498, P=2.73х10 -8 ). Linkage disequilibrium analysis confirmed linc-NFE2L3-1 harbors stronger GWAS signals than protein-coding genes in the locus, suggesting lincRNA might be causal for GWAS association with BMI. Bioinformatic prediction algorithms identified potential binding sites in linc-DMRT2 and linc-NFE2L3-1 for multiple microRNAs that have been demonstrated to regulate adipogenesis (e.g. miR-15 a/b and let-7) or adipocyte functions (e.g. miR-320). In summary, our data suggest that cytoplasmic linc-DMRT2 and linc-NFE2L3-1 may play important roles in adipocyte biology by functioning as competing endogenous RNAs and binding specific microRNAs that mediate adipocyte cellular functions. Genetic variation in such human linRNAs may contribute to cardiometabolic traits.

scholarly journals Munc18-1 is a molecular chaperone for α-synuclein, controlling its self-replicating aggregation

2016 ◽  
Vol 214 (6) ◽  
pp. 705-718 ◽  
Author(s):  
Ye Jin Chai ◽  
Emma Sierecki ◽  
Vanesa M. Tomatis ◽  
Rachel S. Gormal ◽  
Nichole Giles ◽  
...  
Keyword(s):  
Single Molecule ◽  
Molecular Chaperone ◽  
Neurotransmitter Release ◽  
Lewy Body ◽  
Epileptic Encephalopathy ◽  
Chaperone Activity ◽  
Wild Type ◽  
Developmental Defects ◽  
Single Molecule Analysis

Munc18-1 is a key component of the exocytic machinery that controls neurotransmitter release. Munc18-1 heterozygous mutations cause developmental defects and epileptic phenotypes, including infantile epileptic encephalopathy (EIEE), suggestive of a gain of pathological function. Here, we used single-molecule analysis, gene-edited cells, and neurons to demonstrate that Munc18-1 EIEE-causing mutants form large polymers that coaggregate wild-type Munc18-1 in vitro and in cells. Surprisingly, Munc18-1 EIEE mutants also form Lewy body–like structures that contain α-synuclein (α-Syn). We reveal that Munc18-1 binds α-Syn, and its EIEE mutants coaggregate α-Syn. Likewise, removal of endogenous Munc18-1 increases the aggregative propensity of α-SynWT and that of the Parkinson’s disease–causing α-SynA30P mutant, an effect rescued by Munc18-1WT expression, indicative of chaperone activity. Coexpression of the α-SynA30P mutant with Munc18-1 reduced the number of α-SynA30P aggregates. Munc18-1 mutations and haploinsufficiency may therefore trigger a pathogenic gain of function through both the corruption of native Munc18-1 and a perturbed chaperone activity for α-Syn leading to aggregation-induced neurodegeneration.

scholarly journals Conserved roles of RECQ-like helicases Sgs1 and BLM in preventing R-loop associated genome instability

2017 ◽  
Author(s):  
Carolina A. Novoa ◽  
Emily Yun-Chia Chang ◽  
Maria J. Aristizabal ◽  
Yan Coulombe ◽  
Romulo Segovia ◽  
...  
Keyword(s):  
Genome Instability ◽  
Human Cancer ◽  
Wide Distribution ◽  
Dna Helicase ◽  
Human Cancer Cells ◽  
Genome Wide ◽  
Blm Helicase ◽  
Dna Replication And Repair ◽  
Copy Number Changes

AbstractSgs1 is a yeast DNA helicase functioning in DNA replication and repair, and is the orthologue of the human Bloom’s syndrome helicase BLM. Here we analyze the mutation signature associated with SGS1 deletion in yeast, and find frequent copy number changes flanked by regions of repetitive sequence and high R-loop forming potential. We show that loss of SGS1 increases R-loop accumulation and sensitizes cells to replication-transcription collisions. Accordingly, in sgs1Δ cells the genome-wide distribution of R-loops shifts to known sites of Sgs1 action, replication pausing regions, and to long genes. Depletion of the orthologous BLM helicase from human cancer cells also increases R-loop levels, and R-loop-associated genome instability. In support of a direct effect, BLM is found physically proximal to DNA:RNA hybrids in human cells, and can efficiently unwind R-loops in vitro. Together our data describe a conserved role for Sgs1/BLM in R-loop suppression and support an increasingly broad view of DNA repair and replication fork stabilizing proteins as modulators of R-loop mediated genome instability.

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