scholarly journals Poly(ADP-Ribosyl) Glycohydrolase Prevents the Accumulation of Unusual Replication Structures during Unperturbed S Phase

2014 ◽  
Vol 35 (5) ◽  
pp. 856-865 ◽  
Author(s):  
Arnab Ray Chaudhuri ◽  
Akshay Kumar Ahuja ◽  
Raquel Herrador ◽  
Massimo Lopes
Keyword(s):  
Cellular Response ◽  
Genome Stability ◽  
Microscopic Analysis ◽  
Strand Break ◽  
Replication Forks ◽  
Chromosomal Breakage ◽  
Electron Microscopic ◽  
Marked Accumulation ◽  
Chemotherapeutic Treatment ◽  
Replication Intermediates

Poly(ADP-ribosyl)ation (PAR) has been implicated in various aspects of the cellular response to DNA damage and genome stability. Although 17 human poly(ADP-ribose) polymerase (PARP) genes have been identified, a single poly(ADP-ribosyl) glycohydrolase (PARG) mediates PAR degradation. Here we investigated the role of PARG in the replication of human chromosomes. We show that PARG depletion affects cell proliferation and DNA synthesis, leading to replication-coupled H2AX phosphorylation. Furthermore, PARG depletion or inhibitionper seslows down individual replication forks similarly to mild chemotherapeutic treatment. Electron microscopic analysis of replication intermediates reveals marked accumulation of reversed forks and single-stranded DNA (ssDNA) gaps in unperturbed PARG-defective cells. Intriguingly, while we found no physical evidence for chromosomal breakage, PARG-defective cells displayed both ataxia-telangiectasia-mutated (ATM) and ataxia-Rad3-related (ATR) activation, as well as chromatin recruitment of standard double-strand-break-repair factors, such as 53BP1 and RAD51. Overall, these data prove PAR degradation to be essential to promote resumption of replication at endogenous and exogenous lesions, preventing idle recruitment of repair factors to remodeled replication forks. Furthermore, they suggest that fork remodeling and restarting are surprisingly frequent in unperturbed cells and provide a molecular rationale to explore PARG inhibition in cancer chemotherapy.

Control of genome stability by Slx protein complexes

2009 ◽  
Vol 37 (3) ◽  
pp. 495-510 ◽  
Author(s):  
John Rouse
Keyword(s):  
Dna Damage ◽  
Dna Replication ◽  
Protein Interactions ◽  
Cellular Response ◽  
Genome Stability ◽  
Molecular Mechanisms ◽  
Excision Repair ◽  
Protein Complexes ◽  
Alkylating Agents ◽  
Strand Break

The six Saccharomyces cerevisiae SLX genes were identified in a screen for factors required for the viability of cells lacking Sgs1, a member of the RecQ helicase family involved in processing stalled replisomes and in the maintenance of genome stability. The six SLX gene products form three distinct heterodimeric complexes, and all three have catalytic activity. Slx3–Slx2 (also known as Mus81–Mms4) and Slx1–Slx4 are both heterodimeric endonucleases with a marked specificity for branched replication fork-like DNA species, whereas Slx5–Slx8 is a SUMO (small ubiquitin-related modifier)-targeted E3 ubiquitin ligase. All three complexes play important, but distinct, roles in different aspects of the cellular response to DNA damage and perturbed DNA replication. Slx4 interacts physically not only with Slx1, but also with Rad1–Rad10 [XPF (xeroderma pigmentosum complementation group F)–ERCC1 (excision repair cross-complementing 1) in humans], another structure-specific endonuclease that participates in the repair of UV-induced DNA damage and in a subpathway of recombinational DNA DSB (double-strand break) repair. Curiously, Slx4 is essential for repair of DSBs by Rad1–Rad10, but is not required for repair of UV damage. Slx4 also promotes cellular resistance to DNA-alkylating agents that block the progression of replisomes during DNA replication, by facilitating the error-free mode of lesion bypass. This does not require Slx1 or Rad1–Rad10, and so Slx4 has several distinct roles in protecting genome stability. In the present article, I provide an overview of our current understanding of the cellular roles of the Slx proteins, paying particular attention to the advances that have been made in understanding the cellular roles of Slx4. In particular, protein–protein interactions and underlying molecular mechanisms are discussed and I draw attention to the many questions that have yet to be answered.

scholarly journals 53BP1 Mediates ATR-Chk1 Signaling and Protects Replication Forks under Conditions of Replication Stress

2018 ◽  
Vol 38 (8) ◽  
Author(s):  
Joonyoung Her ◽  
Chandni Ray ◽  
Jake Altshuler ◽  
Haiyan Zheng ◽  
Samuel F. Bunting
Keyword(s):  
Cell Death ◽  
Cellular Response ◽  
Ex Vivo ◽  
Replication Stress ◽  
Strand Break ◽  
Replication Forks ◽  
P53 Signaling ◽  
Dna Double Strand Break ◽  
Short Term Exposure ◽  
Stress Signal

ABSTRACTComplete replication of the genome is an essential prerequisite for normal cell division, but a variety of factors can block the replisome, triggering replication stress and potentially causing mutation or cell death. The cellular response to replication stress involves recruitment of proteins to stabilize the replication fork and transmit a stress signal to pause the cell cycle and allow fork restart. We find that the ubiquitously expressed DNA damage response factor 53BP1 is required for the normal response to replication stress. Using primary,ex vivoB cells, we showed that a population of 53BP1−/−cells in early S phase is hypersensitive to short-term exposure to three different agents that induce replication stress. 53BP1 localizes to a subset of replication forks following induced replication stress, and an absence of 53BP1 leads to defective ATR-Chk1-p53 signaling and caspase 3-mediated cell death. Nascent replicated DNA additionally undergoes degradation in 53BP1−/−cells. These results show that 53BP1 plays an important role in protecting replication forks during the cellular response to replication stress, in addition to the previously characterized role of 53BP1 in DNA double-strand break repair.

scholarly journals Replication Restart in Bacteria

2017 ◽  
Vol 199 (13) ◽  
Author(s):  
Bénédicte Michel ◽  
Steven J. Sandler
Keyword(s):  
Homologous Recombination ◽  
Genome Stability ◽  
Strand Break ◽  
Replication Initiation ◽  
Replication Forks ◽  
Independent Manner ◽  
Close Link ◽  
Replication Restart

ABSTRACT In bacteria, replication forks assembled at a replication origin travel to the terminus, often a few megabases away. They may encounter obstacles that trigger replisome disassembly, rendering replication restart from abandoned forks crucial for cell viability. During the past 25 years, the genes that encode replication restart proteins have been identified and genetically characterized. In parallel, the enzymes were purified and analyzed in vitro, where they can catalyze replication initiation in a sequence-independent manner from fork-like DNA structures. This work also revealed a close link between replication and homologous recombination, as replication restart from recombination intermediates is an essential step of DNA double-strand break repair in bacteria and, conversely, arrested replication forks can be acted upon by recombination proteins and converted into various recombination substrates. In this review, we summarize this intense period of research that led to the characterization of the ubiquitous replication restart protein PriA and its partners, to the definition of several replication restart pathways in vivo, and to the description of tight links between replication and homologous recombination, responsible for the importance of replication restart in the maintenance of genome stability.

scholarly journals The sep1 mutant of Saccharomyces cerevisiae arrests in pachytene and is deficient in meiotic recombination.

Genetics ◽  
1995 ◽  
Vol 139 (2) ◽  
pp. 495-509 ◽  
Author(s):  
D X Tishkoff ◽  
B Rockmill ◽  
G S Roeder ◽  
R D Kolodner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Meiotic Recombination ◽  
Microscopic Analysis ◽  
Mutant Gene ◽  
Strand Break ◽  
Strand Exchange ◽  
Wild Type ◽  
Electron Microscopic ◽  
Pathways Analysis

Abstract Strand exchange protein 1 (Sep1) from Saccharomyces cerevisiae promotes homologous pairing of DNA in vitro and sep1 mutants display pleiotropic phenotypes in both vegetative and meiotic cells. In this study, we examined in detail the ability of the sep1 mutant to progress through meiosis I prophase and to undergo meiotic recombination. In meiotic return-to-growth experiments, commitment to meiotic recombination began at the same time in wild type and mutant; however, recombinants accumulated at decreased rates in the mutant. Gene conversion eventually reached nearly wild-type levels, whereas crossing over reached 15-50% of wild type. In an assay of intrachromosomal pop-out recombination, the sep1, dmc1 and rad51 single mutations had only small effects; however, pop-out recombination was virtually eliminated in the sep1 dmc1 and sep1 rad51 double mutants, providing evidence for multiple recombination pathways. Analysis of meiotic recombination intermediates indicates that the sep1 mutant is deficient in meiotic double-strand break repair. In a physical assay, the formation of mature reciprocal recombinants in the sep1 mutant was delayed relative to wild type and ultimately reached only 50% of the wild-type level. Electron microscopic analysis of meiotic nuclear spreads indicates that the sep1 delta mutant arrests in pachytene, with apparently normal synaptonemal complex. This arrest is RAD9-independent. We hypothesize that the Sep1 protein participates directly in meiotic recombination and that other strand exchange enzymes, acting in parallel recombination pathways, are able to substitute partially for the absence of the Sep1 protein.

scholarly journals Electron microscopic analysis of rotavirus assembly-replication intermediates

Virology ◽  
2015 ◽  
Vol 477 ◽  
pp. 32-41 ◽  
Author(s):  
Crystal E. Boudreaux ◽  
Deborah F. Kelly ◽  
Sarah M. McDonald
Keyword(s):  
Microscopic Analysis ◽  
Electron Microscopic Analysis ◽  
Electron Microscopic ◽  
Replication Intermediates

scholarly journals Interferon-stimulated gene 15 accelerates replication fork progression inducing chromosomal breakage

2020 ◽  
Vol 219 (8) ◽  
Author(s):  
Maria Chiara Raso ◽  
Nikola Djoric ◽  
Franziska Walser ◽  
Sandra Hess ◽  
Fabian Marc Schmid ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Replication ◽  
Replication Fork ◽  
Genome Stability ◽  
Dna Helicase ◽  
Replication Forks ◽  
Chromosomal Breakage ◽  
Replication Fork Progression ◽  
Ubiquitin System ◽  
Interferon Stimulated Gene 15

DNA replication is highly regulated by the ubiquitin system, which plays key roles upon stress. The ubiquitin-like modifier ISG15 (interferon-stimulated gene 15) is induced by interferons, bacterial and viral infection, and DNA damage, but it is also constitutively expressed in many types of cancer, although its role in tumorigenesis is still largely elusive. Here, we show that ISG15 localizes at the replication forks, in complex with PCNA and the nascent DNA, where it regulates DNA synthesis. Indeed, high levels of ISG15, intrinsic or induced by interferon-β, accelerate DNA replication fork progression, resulting in extensive DNA damage and chromosomal aberrations. This effect is largely independent of ISG15 conjugation and relies on ISG15 functional interaction with the DNA helicase RECQ1, which promotes restart of stalled replication forks. Additionally, elevated ISG15 levels sensitize cells to cancer chemotherapeutic treatments. We propose that ISG15 up-regulation exposes cells to replication stress, impacting genome stability and response to genotoxic drugs.

scholarly journals Never cared for what they do. High structural stability of Guanine-quadruplexes in presence of strand-break damages

2021 ◽  
Author(s):  
Tom Miclot ◽  
Cécilia Hognon ◽  
Emmanuelle Bignon ◽  
Alessio Terenzi ◽  
Stéphanie Grandemange ◽  
...  
Keyword(s):  
Structural Stability ◽  
Cellular Response ◽  
Genome Stability ◽  
Strand Break ◽  
Dna Lesions ◽  
Dna Integrity ◽  
Repair System ◽  
Strand Breaks ◽  
G4 Dna ◽  
Complete Restoration

AbstractDNA integrity is an important factor to assure genome stability and, more generally, cells and organisms’ viability. In presence of DNA damage, the normal cell cycle is perturbed while cells activate their repair processes. Although efficient, the repair system is not always able to ensure the complete restoration of gene integrity. In these cases, not only mutations may occur, but the accumulation of lesions can either lead to carcinogenesis or reach a threshold which induces apoptosis and the programmed cell death. Among the different types of DNA lesions, strand breaks produced by ionizing radiations are the most toxic, due to their inherently difficult repair, which may lead to genomic instability. In this article we show, by using classical molecular simulations techniques, that differently from the canonical double-helical B-DNA, guanine-quadruplex (G4) arrangements show a remarkable structural stability, even in presence of two strand breaks. Since G4-DNA are recognized for their regulatory roles in cell senescence and gene expression, also involving oncogene, their stability can be related to an evolutionary cellular response aimed at minimizing the effects of ionizing radiation.

Preparatory Procedures for Electron Microscopic Analysis at the Molecular Level of Resolution

1978 ◽  
Vol 36 (3) ◽  
pp. 516-520
Author(s):  
F.J. Sjostrand
Keyword(s):  
Electron Microscope ◽  
Molecular Level ◽  
Microscopic Analysis ◽  
Resolving Power ◽  
Cellular Structures ◽  
Electron Microscopic ◽  
Systematic Analysis ◽  
Technical Developments ◽  
Thin Sectioning ◽  
Obvious Reason

In the 1940's and 1950's electron microscopy conferences were attended with everybody interested in learning about the latest technical developments for one very obvious reason. There was the electron microscope with its outstanding performance but nobody could make very much use of it because we were lacking proper techniques to prepare biological specimens. The development of the thin sectioning technique with its perfectioning in 1952 changed the situation and systematic analysis of the structure of cells could now be pursued. Since then electron microscopists have in general become satisfied with the level of resolution at which cellular structures can be analyzed when applying this technique. There has been little interest in trying to push the limit of resolution closer to that determined by the resolving power of the electron microscope.

Electron Microscopic Analysis of Myonecrosis Induced by a Pure Component Isolated From Rattlesnake Venom

1976 ◽  
Vol 34 ◽  
pp. 292-293
Author(s):  
Charlotte L. Ownby ◽  
David Cameron ◽  
Anthony T. Tu
Keyword(s):  
Gel Filtration ◽  
Microscopic Analysis ◽  
The United States ◽  
Exchange Chromatography ◽  
Pure Component ◽  
Electron Microscopic Level ◽  
Electron Microscopic ◽  
Mouse Muscle ◽  
Major Health Problem ◽  
Rattlesnake Venom

In the United States the major health problem resulting from snakebite poisoning is local tissue damage, i.e. hemorrhage and myonecrosis. Since commercial antivenin does not usually prevent such damage to tissue, a more effective treatment of snakebite-induced myonecrosis is needed. To aid in the development of such a treatment the pathogenesis of myonecrosis induced by a pure component of rattlesnake venom was studied at the electron microscopic level.The pure component, a small (4,300 mol. wt.), basic (isoelectric point of 9.6) protein, was isolated from crude prairie rattlesnake (Crotalus viridis viridis) venom by gel filtration (Sephadex G-50) followed by cation exchange chromatography (Sephadex C-25), and shown to be pure by electrophoresis. Selection of the myotoxic component was based on light microscopic observations of injected mouse muscle.

Correlative video enhanced differential interference contrast light microscopy (VDIC)-LM), low-voltage high-resolution SEM (LV-HR-SEM), and high-voltage electron microscopy (HVEM) in the observation of gold-labelled surface antigens and the subjacent cytoskeletal matrix

1989 ◽  
Vol 47 ◽  
pp. 904-905
Author(s):  
Ralph M. Albrecht ◽  
Scott R. Simmons ◽  
Marek Malecki
Keyword(s):  
High Resolution ◽  
Light Microscopy ◽  
Low Voltage ◽  
Microscopic Analysis ◽  
Cell Labelling ◽  
Video Technology ◽  
Electron Microscopic ◽  
Image Capture ◽  
High Voltage Electron Microscopy ◽  
Fluorescence Studies

The development of video-enhanced light microscopy (LM) as well as associated image processing and analysis have significantly broadened the scope of investigations which can be undertaken using (LM). Interference/polarization based microscopies can provide high resolution and higher levels of “detectability” especially in unstained living systems. Confocal light microscopy also holds the promise of further improvements in resolution, fluorescence studies, and 3 dimensional reconstruction. Video technology now provides, among other things, a means to detect differences in contrast difficult to detect with the human eye; furthermore, computerized image capture, processing, and analysis can be used to enhance features of interest, average images, subtract background, and provide a quantitative basis to studies of cells, cell features, cell labelling, and so forth. Improvements in video technology, image capture, and cost-effective computer image analysis/processing have contributed to the utility and potential of the various interference and confocal microscopic instrumentation.Electron microscopic technology has made advances as well. Microprocessor control and improved design have contributed to high resolution SEMs which have imaging capability at the molecular level and can operate at a range of accelerating voltages starting at 1KV. Improvements have also been seen in the HVEM and IVEM transmission instruments. As a whole, these advances in LM and EM microscopic technology provide the biologist with an array of information on structure, composition, and function which can be obtained from a single specimen. Corrrelative light microscopic analysis permits examination of living specimens and is critical where the “history” of a cell, cellular components, or labels needs to be known up to the time of chemical or physical fixation. Features such as cytoskeletal elements or gold label as small as 0.01 μm, well below the 0.2 μm limits of LM resolution, can be “detected” and their movement followed by VDIC-LM. Appropriate identification and preparation can then lead to the examination of surface detail and surface label with stereo LV-HR-SEM. Increasing the KV in the HR-SEM while viewing uncoated or thinly coated specimens can provide information from beneath the surface as well as increasing Z contrast so that positive identification of surface and subsurface colloidal gold or other heavy metal labelled/stained material is possible. Further examination of the same cells using stereo HVEM or IVEM provides information on internal ultrastructure and on the relationship of labelled material to cytoskeletal or organellar distribution, A wide variety of investigations can benefit from this correlative approach and a number of instrumentational configurations and preparative pathways can be tailored for the particular study. For a surprisingly small investment in time and technique, it is often possible to clear ambiguities or questions that arise when a finding is presented in the context of only one modality.

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