scholarly journals Quantitative methods to investigate the 4D dynamics of heterochromatic repair sites in Drosophila cells

2017 ◽  
Author(s):  
Christopher P. Caridi ◽  
Laetitia Delabaere ◽  
Harianto Tjong ◽  
Hannah Hopp ◽  
Devika Das ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Genome Stability ◽  
Quantitative Methods ◽  
Nuclear Periphery ◽  
Three Dimensional ◽  
Mean Square Displacement ◽  
Strand Break ◽  
Signal Recovery ◽  
Nuclear Dynamics ◽  
Fluorescent Markers

AbstractHeterochromatin is mostly composed of long stretches of repeated DNA sequences prone to ectopic recombination during double-strand break (DSB) repair. In Drosophila, ‘safe’ homologous recombination (HR) repair of heterochromatic DSBs relies on a striking relocalization of repair sites to the nuclear periphery. Central to understanding heterochromatin repair is the ability to investigate the 4D dynamics (movement in space and time) of repair sites. A specific challenge of these studies is preventing phototoxicity and photobleaching effects while imaging the sample over long periods of time, and with sufficient time points and Z-stacks to track repair foci over time. Here we describe an optimized approach for high-resolution live imaging of heterochromatic DSBs in Drosophila cells, with a specific emphasis on the fluorescent markers and imaging setup used to capture the motion of repair foci over long time periods. We detail approaches that minimize photobleaching and phototoxicity with a DeltaVision widefield deconvolution microscope, and image-processing techniques for signal recovery post-imaging using SoftWorX and Imaris software. We present a method to derive mean square displacement (MSD) curves revealing some of the biophysical properties of the motion. Finally, describe a method in R to identify tracts of directed motions in mixed trajectories. These approaches enable a deeper understanding of the mechanisms of heterochromatin dynamics and genome stability in the three-dimensional context of the nucleus, and have broad applicability in the field of nuclear dynamics.

scholarly journals Arp2/3 and Unc45 maintain heterochromatin stability in Drosophila polytene chromosomes

2019 ◽  
Author(s):  
George Dialynas ◽  
Laetitia Delabaere ◽  
Irene Chiolo
Keyword(s):  
Actin Filaments ◽  
Genome Stability ◽  
Actin Polymerization ◽  
Cultured Cells ◽  
Nuclear Periphery ◽  
Polytene Chromosomes ◽  
Nuclear Actin ◽  
Nuclear Dynamics ◽  
Ectopic Recombination ◽  
Repair Pathways

AbstractRepairing DNA double-strand breaks (DSBs) is particularly challenging in pericentromeric heterochromatin, where the abundance of repeated sequences exacerbates the risk of ectopic recombination. InDrosophilaKc cells, accurate homologous recombination (HR) repair of heterochromatic DSBs relies on the relocalization of repair sites to the nuclear periphery before Rad51 recruitment and strand invasion. This movement is driven by Arp2/3-dependent nuclear actin filaments and myosins’ ability to walk along them. Conserved mechanisms enable the relocalization of heterochromatic DSBs in mouse cells, and their defects lead to massive ectopic recombination in heterochromatin and chromosome rearrangements. InDrosophilapolytene chromosomes, extensive DNA movement is blocked by a stiff structure of chromosome bundles. Repair pathways in this context are poorly characterized, and whether heterochromatic DSBs relocalize in these cells is unknown. Here, we show that damage in heterochromatin results in relaxation of the heterochromatic chromocenter, consistent with a dynamic response in this structure. Arp2/3, the Arp2/3 activator Scar, and the myosin activator Unc45, are required for heterochromatin stability in polytene cells, suggesting that relocalization enables heterochromatin repair in this tissue. Together, these studies reveal critical roles for actin polymerization and myosin motors in heterochromatin repair and genome stability across different organisms and tissue types.Impact StatementHeterochromatin relies on dedicated pathways for ‘safe’ recombinational repair. In mouse and fly cultured cells, DNA repair requires the movement of repair sites away from the heterochromatin ‘domain’vianuclear actin filaments and myosins. Here, we explore the importance of these pathways inDrosophilasalivary gland cells, which feature a stiff bundle of endoreduplicated polytene chromosomes. Repair pathways in polytene chromosomes are largely obscure and how nuclear dynamics operate in this context is unknown. We show that heterochromatin relaxes in response to damage, and relocalization pathways are necessary for repair and stability of heterochromatic sequences. This deepens our understanding of repair mechanisms in polytenes, revealing unexpected dynamics. It also provides a first understanding of nuclear dynamics responding to replication damage or rDNA breaks, providing a new understanding of the importance of the nucleoskeleton in genome stability. We expect these discoveries to shed light on tumorigenic processes, including therapy-induced cancer relapses.

scholarly journals And yet, it moves: nuclear and chromatin dynamics of a heterochromatic double-strand break

2017 ◽  
Vol 372 (1731) ◽  
pp. 20160291 ◽  
Author(s):  
P. Christopher Caridi ◽  
Laetitia Delabaere ◽  
Grzegorz Zapotoczny ◽  
Irene Chiolo
Keyword(s):  
Dna Sequences ◽  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Nuclear Periphery ◽  
Strand Break ◽  
Double Strand Break ◽  
Chromatin Dynamics ◽  
Recombination Repair ◽  
The Stability

Heterochromatin is mostly composed of repeated DNA sequences prone to aberrant recombination. How cells maintain the stability of these sequences during double-strand break (DSB) repair has been a long-standing mystery. Studies in Drosophila cells revealed that faithful homologous recombination repair of heterochromatic DSBs relies on the striking relocalization of repair sites to the nuclear periphery before Rad51 recruitment and repair progression. Here, we summarize our current understanding of this response, including the molecular mechanisms involved, and conserved pathways in mammalian cells. We will highlight important similarities with pathways identified in budding yeast for repair of other types of repeated sequences, including rDNA and short telomeres. We will also discuss the emerging role of chromatin composition and regulation in heterochromatin repair progression. Together, these discoveries challenged previous assumptions that repair sites are substantially static in multicellular eukaryotes, that heterochromatin is largely inert in the presence of DSBs, and that silencing and compaction in this domain are obstacles to repair. This article is part of the themed issue ‘Chromatin modifiers and remodellers in DNA repair and signalling’.

scholarly journals A decade of understanding spatio-temporal regulation of DNA repair by the nuclear architecture

2016 ◽  
Vol 94 (5) ◽  
pp. 433-440 ◽  
Author(s):  
Hicham Saad ◽  
Jennifer A. Cobb
Keyword(s):  
Dna Repair ◽  
Genome Stability ◽  
Nuclear Organization ◽  
Chromosomal Rearrangements ◽  
Nuclear Periphery ◽  
Nuclear Architecture ◽  
Strand Break ◽  
Repair Pathway ◽  
Pathway Choice ◽  
Spatio Temporal

The nucleus is a hub for gene expression and is a highly organized entity. The nucleoplasm is heterogeneous, owing to the preferential localization of specific metabolic factors, which lead to the definition of nuclear compartments or bodies. The genome is organized into chromosome territories, as well as heterochromatin and euchromatin domains. Recent observations have indicated that nuclear organization is important for maintaining genomic stability. For example, nuclear organization has been implicated in stabilizing damaged DNA, repair-pathway choice, and in preventing chromosomal rearrangements. Over the past decade, several studies have revealed that dynamic changes in the nuclear architecture are important during double-strand break repair. Stemming from work in yeast, relocation of a damaged site prior to repair appears to be at least partially conserved in multicellular eukaryotes. In this review, we will discuss genome and nucleoplasm architecture, particularly the importance of the nuclear periphery in genome stability. We will also discuss how the site of relocation regulates repair-pathway choice.

Three-Dimensional Structure of Cloned T3 Connector Protein at 1.6nm Resolution

1990 ◽  
Vol 48 (1) ◽  
pp. 282-283
Author(s):  
José L. Carrascosa ◽  
José M. Valpuesta ◽  
Hisao Fujisawa
Keyword(s):  
Dna Sequences ◽  
Expression Vector ◽  
Three Dimensional ◽  
Deae Cellulose ◽  
Dna Packaging ◽  
Dimensional Structure ◽  
Two Dimensional ◽  
Freezing And Thawing ◽  
Three Dimensional Structure ◽  
Dimensional Reconstruction

The head to tail connector of bacteriophages plays a fundamental role in the assembly of viral heads and DNA packaging. In spite of the absence of sequence homology, the structure of connectors from different viruses (T4, Ø29, T3, P22, etc) share common morphological features, that are most clearly revealed in their three-dimensional structure. We have studied the three-dimensional reconstruction of the connector protein from phage T3 (gp 8) from tilted view of two dimensional crystals obtained from this protein after cloning and purification.DNA sequences including gene 8 from phage T3 were cloned, into Bam Hl-Eco Rl sites down stream of lambda promotor PL, in the expression vector pNT45 under the control of cI857. E R204 (pNT89) cells were incubated at 42°C for 2h, harvested and resuspended in 20 mM Tris HC1 (pH 7.4), 7mM 2 mercaptoethanol, ImM EDTA. The cells were lysed by freezing and thawing in the presence of lysozyme (lmg/ml) and ligthly sonicated. The low speed supernatant was precipitated by ammonium sulfate (60% saturated) and dissolved in the original buffer to be subjected to gel nitration through Sepharose 6B, followed by phosphocellulose colum (Pll) and DEAE cellulose colum (DE52). Purified gp8 appeared at 0.3M NaCl and formed crystals when its concentration increased above 1.5 mg/ml.

scholarly journals ATRX promotes heterochromatin formation to protect cells from G-quadruplex DNA-mediated stress

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Yu-Ching Teng ◽  
Aishwarya Sundaresan ◽  
Ryan O’Hara ◽  
Vincent U. Gant ◽  
Minhua Li ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Genome Stability ◽  
Helicase Domain ◽  
Quadruplex Dna ◽  
Heterochromatin Formation ◽  
Replicative Stress ◽  
G4 Dna ◽  
Mutation Burden ◽  
G Quadruplex ◽  
Dna Replication Stress

AbstractATRX is a tumor suppressor that has been associated with protection from DNA replication stress, purportedly through resolution of difficult-to-replicate G-quadruplex (G4) DNA structures. While several studies demonstrate that loss of ATRX sensitizes cells to chemical stabilizers of G4 structures, the molecular function of ATRX at G4 regions during replication remains unknown. Here, we demonstrate that ATRX associates with a number of the MCM replication complex subunits and that loss of ATRX leads to G4 structure accumulation at newly synthesized DNA. We show that both the helicase domain of ATRX and its H3.3 chaperone function are required to protect cells from G4-induced replicative stress. Furthermore, these activities are upstream of heterochromatin formation mediated by the histone methyltransferase, ESET, which is the critical molecular event that protects cells from G4-mediated stress. In support, tumors carrying mutations in either ATRX or ESET show increased mutation burden at G4-enriched DNA sequences. Overall, our study provides new insights into mechanisms by which ATRX promotes genome stability with important implications for understanding impacts of its loss on human disease.

scholarly journals RAD6 Promotes Homologous Recombination Repair by Activating the Autophagy-Mediated Degradation of Heterochromatin Protein HP1

2014 ◽  
Vol 35 (2) ◽  
pp. 406-416 ◽  
Author(s):  
Su Chen ◽  
Chen Wang ◽  
Luxi Sun ◽  
Da-Liang Wang ◽  
Lu Chen ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Genome Stability ◽  
Chromosomal Rearrangements ◽  
Strand Break ◽  
Open Chromatin ◽  
Dsb Repair ◽  
Heterochromatin Protein ◽  
Dna Double Strand Break ◽  
Recombination Repair ◽  
Ubiquitin Conjugating Enzyme

Efficient DNA double-strand break (DSB) repair is critical for the maintenance of genome stability. Unrepaired or misrepaired DSBs cause chromosomal rearrangements that can result in severe consequences, such as tumorigenesis. RAD6 is an E2 ubiquitin-conjugating enzyme that plays a pivotal role in repairing UV-induced DNA damage. Here, we present evidence that RAD6 is also required for DNA DSB repair via homologous recombination (HR) by specifically regulating the degradation of heterochromatin protein 1α (HP1α). Our study indicates that RAD6 physically interacts with HP1α and ubiquitinates HP1α at residue K154, thereby promoting HP1α degradation through the autophagy pathway and eventually leading to an open chromatin structure that facilitates efficient HR DSB repair. Furthermore, bioinformatics studies have indicated that the expression of RAD6 and HP1α exhibits an inverse relationship and correlates with the survival rate of patients.

scholarly journals Site Specific Three-dimensional Structural Analysis in Tissues and Cells Using Automated DualBeam Slice &View

2007 ◽  
Vol 15 (2) ◽  
pp. 26-31 ◽  
Author(s):  
Ben Lich
Keyword(s):  
Electron Microscopy ◽  
Focused Ion Beam ◽  
Ion Beam ◽  
Three Dimensional ◽  
Surface Region ◽  
Dimensional Structure ◽  
Near Surface ◽  
Imaging Capability ◽  
Fluorescent Markers ◽  
Confocal And Electron Microscopy

DualBeam instruments that combine the imaging capability of scanning electron microscopy (SEM) with the cutting and deposition capability of a focused ion beam (FIB) provide biologists with a powerful tool for investigating three-dimensional structure with nanoscale (1 nm-100 nm) resolution. Ever since Van Leeuwenhoek used the first microscope to describe bacteria more than 300 years ago, microscopy has played a central role in scientists' efforts to understand biological systems. Light microscopy is generally limited to a useful resolution of about a micrometer. More recently the use of confocal and electron microscopy has enabled investigations at higher resolution. Used with fluorescent markers, confocal microscopy can detect and localize molecular scale features, but its imaging resolution is still limited. SEM is capable of nanometer resolution, but is limited to the near surface region of the sample.

scholarly journals Shelterin and subtelomeric DNA sequences control nucleosome maintenance and genome stability

EMBO Reports ◽  
2018 ◽  
Vol 20 (1) ◽  
Author(s):  
Thomas S Emden ◽  
Marta Forn ◽  
Ignasi Forné ◽  
Zsuzsa Sarkadi ◽  
Matías Capella ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Genome Stability

scholarly journals i-BLESS is an ultra-sensitive method for detection of DNA double-strand breaks

2018 ◽  
Vol 1 (1) ◽  
Author(s):  
Anna Biernacka ◽  
Yingjie Zhu ◽  
Magdalena Skrzypczak ◽  
Romain Forey ◽  
Benjamin Pardo ◽  
...  
Keyword(s):  
Cell Fate ◽  
Genome Stability ◽  
Strand Break ◽  
Double Strand Breaks ◽  
Universal Method ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Agarose Beads ◽  
Genome Wide ◽  
Dna Double Strand Break

AbstractMaintenance of genome stability is a key issue for cell fate that could be compromised by chromosome deletions and translocations caused by DNA double-strand breaks (DSBs). Thus development of precise and sensitive tools for DSBs labeling is of great importance for understanding mechanisms of DSB formation, their sensing and repair. Until now there has been no high resolution and specific DSB detection technique that would be applicable to any cells regardless of their size. Here, we present i-BLESS, a universal method for direct genome-wide DNA double-strand break labeling in cells immobilized in agarose beads. i-BLESS has three key advantages: it is the only unbiased method applicable to yeast, achieves a sensitivity of one break at a given position in 100,000 cells, and eliminates background noise while still allowing for fixation of samples. The method allows detection of ultra-rare breaks such as those forming spontaneously at G-quadruplexes.

scholarly journals Heterogeneous chromatin mobility derived from chromatin states is a determinant of genome organisation in S. cerevisiae

2017 ◽  
Author(s):  
Sven A. Sewitz ◽  
Zahra Fahmi ◽  
Latifa Aljebali ◽  
Jeremy Bancroft ◽  
Otávio J. B. Brustolini ◽  
...  
Keyword(s):  
Heat Shock ◽  
Spatial Clustering ◽  
Genome Structure ◽  
Nuclear Periphery ◽  
Three Dimensional ◽  
Normal Growth ◽  
Heterogeneous Distribution ◽  
Chromatin States ◽  
Protein Occupancy ◽  
Self Organisation

AbstractSpatial organisation of the genome is essential for regulating gene activity, yet the mechanisms that shape this three-dimensional organisation in eukaryotes are far from understood. Here, we combine bioinformatic determination of chromatin states during normal growth and heat shock, and computational polymer modelling of genome structure, with quantitative microscopy and Hi-C to demonstrate that differential mobility of yeast chromosome segments leads to spatial self-organisation of the genome. We observe that more than forty percent of chromatin-associated proteins display a poised and heterogeneous distribution along the chromosome, creating a heteropolymer. This distribution changes upon heat shock in a concerted, state-specific manner. Simulating yeast chromosomes as heteropolymers, in which the mobility of each segment depends on its cumulative protein occupancy, results in functionally relevant structures, which match our experimental data. This thermodynamically driven self-organisation achieves spatial clustering of poised genes and mechanistically contributes to the directed relocalisation of active genes to the nuclear periphery upon heat shock.One Sentence SummaryUnequal protein occupancy and chromosome segment mobility drive 3D organisation of the genome.

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