scholarly journals The poly(ADP-ribose)-dependent chromatin remodeler Alc1 induces local chromatin relaxation upon DNA damage

2016 ◽  
Vol 27 (24) ◽  
pp. 3791-3799 ◽  
Author(s):  
Hafida Sellou ◽  
Théo Lebeaupin ◽  
Catherine Chapuis ◽  
Rebecca Smith ◽  
Anna Hegele ◽  
...  
Keyword(s):  
Dna Damage ◽  
Structural Changes ◽  
Cellular Responses ◽  
Dna Lesions ◽  
Fast Kinetics ◽  
Important Player ◽  
Chromatin Relaxation ◽  
Kinetics Of ◽  
Laser Microirradiation

Chromatin relaxation is one of the earliest cellular responses to DNA damage. However, what determines these structural changes, including their ATP requirement, is not well understood. Using live-cell imaging and laser microirradiation to induce DNA lesions, we show that the local chromatin relaxation at DNA damage sites is regulated by PARP1 enzymatic activity. We also report that H1 is mobilized at DNA damage sites, but, since this mobilization is largely independent of poly(ADP-ribosyl)ation, it cannot solely explain the chromatin relaxation. Finally, we demonstrate the involvement of Alc1, a poly(ADP-ribose)- and ATP-dependent remodeler, in the chromatin-relaxation process. Deletion of Alc1 impairs chromatin relaxation after DNA damage, while its overexpression strongly enhances relaxation. Altogether our results identify Alc1 as an important player in the fast kinetics of the NAD+- and ATP-dependent chromatin relaxation upon DNA damage in vivo.

scholarly journals Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

10.3791/56213 ◽  
2018 ◽  
Author(s):  
Xiangduo Kong ◽  
Gladys M.S. Cruz ◽  
Bárbara A. Silva ◽  
Nicole M. Wakida ◽  
Nima Khatibzadeh ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cellular Responses ◽  
Laser Microirradiation

scholarly journals Poly(ADP-ribosyl)ation-dependent Transient Chromatin Decondensation and Histone Displacement following Laser Microirradiation

2015 ◽  
Vol 291 (4) ◽  
pp. 1789-1802 ◽  
Author(s):  
Hilmar Strickfaden ◽  
Darin McDonald ◽  
Michael J. Kruhlak ◽  
Jean-Francois Haince ◽  
John P. H. Th'ng ◽  
...  
Keyword(s):  
Dna Damage ◽  
Histone H1 ◽  
Parp Inhibition ◽  
Chromatin Decondensation ◽  
Strand Breaks ◽  
Photoactivatable Gfp ◽  
Dna Strand ◽  
Kinetics Of ◽  
Laser Microirradiation

Chromatin undergoes a rapid ATP-dependent, ATM and H2AX-independent decondensation when DNA damage is introduced by laser microirradiation. Although the detailed mechanism of this decondensation remains to be determined, the kinetics of decondensation are similar to the kinetics of poly(ADP-ribosyl)ation. We used laser microirradiation to introduce DNA strand breaks into living cells expressing a photoactivatable GFP-tagged histone H2B. We find that poly(ADP-ribosyl)ation mediated primarily by poly(ADP-ribose) polymerase 1 (PARP1) is responsible for the rapid decondensation of chromatin at sites of DNA damage. This decondensation of chromatin correlates temporally with the displacement of histones, which is sensitive to PARP inhibition and is transient in nature. Contrary to the predictions of the histone shuttle hypothesis, we did not find that histone H1 accumulated on poly(ADP-ribose) (PAR) in vivo. Rather, histone H1, and to a lessor extent, histones H2A and H2B were rapidly depleted from the sites of PAR accumulation. However, histone H1 returns to chromatin and the chromatin recondenses. Thus, the PARP-dependent relaxation of chromatin closely correlates with histone displacement.

scholarly journals The Zn-finger of Saccharomyces cerevisiae Rad18 and its adjacent region mediate interaction with Rad5

2021 ◽  
Author(s):  
Orsolya Frittmann ◽  
Vamsi K Gali ◽  
Miklos Halmai ◽  
Robert Toth ◽  
Zsuzsanna Gyorfy ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Damage ◽  
Ubiquitin Ligase ◽  
Dna Lesions ◽  
Template Switching ◽  
Adjacent Region ◽  
Yeast Two Hybrid ◽  
Replication Complex ◽  
Two Hybrid

Abstract DNA damages that hinder the movement of the replication complex can ultimately lead to cell death. To avoid that, cells possess several DNA damage bypass mechanisms. The Rad18 ubiquitin ligase controls error-free and mutagenic pathways that help the replication complex to bypass DNA lesions by monoubiquitylating PCNA at stalled replication forks. In Saccharomyces cerevisiae, two of the Rad18 governed pathways are activated by monoubiquitylated PCNA and they involve translesion synthesis polymerases, whereas a third pathway needs subsequent polyubiquitylation of the same PCNA residue by another ubiquitin ligase the Rad5 protein, and it employs template switching. The goal of this study was to dissect the regulatory role of the multidomain Rad18 in DNA damage bypass using a structure-function based approach. Investigating deletion and point mutant RAD18 variants in yeast genetic and yeast two-hybrid assays we show that the Zn-finger of Rad18 mediates its interaction with Rad5, and the N-terminal adjacent region is also necessary for Rad5 binding. Moreover, results of the yeast two-hybrid and in vivo ubiquitylation experiments raise the possibility that direct interaction between Rad18 and Rad5 might not be necessary for the function of the Rad5 dependent pathway. The presented data also reveal that yeast Rad18 uses different domains to mediate its association with itself and with Rad5. Our results contribute to better understanding of the complex machinery of DNA damage bypass pathways.

scholarly journals The splicing factor XAB2 interacts with ERCC1-XPF and XPG for R-loop processing

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Evi Goulielmaki ◽  
Maria Tsekrekou ◽  
Nikos Batsiotos ◽  
Mariana Ascensão-Ferreira ◽  
Eleftheria Ledaki ◽  
...  
Keyword(s):  
Dna Damage ◽  
Rna Splicing ◽  
Excision Repair ◽  
Intron Retention ◽  
Dna Lesions ◽  
Splicing Factor ◽  
Loop Formation ◽  
Rna Targets ◽  
Underlying Mechanisms

AbstractRNA splicing, transcription and the DNA damage response are intriguingly linked in mammals but the underlying mechanisms remain poorly understood. Using an in vivo biotinylation tagging approach in mice, we show that the splicing factor XAB2 interacts with the core spliceosome and that it binds to spliceosomal U4 and U6 snRNAs and pre-mRNAs in developing livers. XAB2 depletion leads to aberrant intron retention, R-loop formation and DNA damage in cells. Studies in illudin S-treated cells and Csbm/m developing livers reveal that transcription-blocking DNA lesions trigger the release of XAB2 from all RNA targets tested. Immunoprecipitation studies reveal that XAB2 interacts with ERCC1-XPF and XPG endonucleases outside nucleotide excision repair and that the trimeric protein complex binds RNA:DNA hybrids under conditions that favor the formation of R-loops. Thus, XAB2 functionally links the spliceosomal response to DNA damage with R-loop processing with important ramifications for transcription-coupled DNA repair disorders.

scholarly journals RuvAB and RecG Are Not Essential for the Recovery of DNA Synthesis Following UV-Induced DNA Damage in Escherichia coli

Genetics ◽  
2004 ◽  
Vol 166 (4) ◽  
pp. 1631-1640 ◽  
Author(s):  
Janet R Donaldson ◽  
Charmain T Courcelle ◽  
Justin Courcelle
Keyword(s):  
Dna Damage ◽  
Dna Synthesis ◽  
Dna Lesions ◽  
Replication Forks ◽  
Wild Type ◽  
Replication Machinery ◽  
Dna Junctions ◽  
Fork Regression

Abstract Ultraviolet light induces DNA lesions that block the progression of the replication machinery. Several models speculate that the resumption of replication following disruption by UV-induced DNA damage requires regression of the nascent DNA or migration of the replication machinery away from the blocking lesion to allow repair or bypass of the lesion to occur. Both RuvAB and RecG catalyze branch migration of three- and four-stranded DNA junctions in vitro and are proposed to catalyze fork regression in vivo. To examine this possibility, we characterized the recovery of DNA synthesis in ruvAB and recG mutants. We found that in the absence of either RecG or RuvAB, arrested replication forks are maintained and DNA synthesis is resumed with kinetics that are similar to those in wild-type cells. The data presented here indicate that RecG- or RuvAB-catalyzed fork regression is not essential for DNA synthesis to resume following arrest by UV-induced DNA damage in vivo.

scholarly journals Interactions and Localization of Escherichia coli Error-Prone DNA Polymerase IV after DNA Damage

2015 ◽  
Vol 197 (17) ◽  
pp. 2792-2809 ◽  
Author(s):  
Sarita Mallik ◽  
Ellen M. Popodi ◽  
Andrew J. Hanson ◽  
Patricia L. Foster
Keyword(s):  
Dna Damage ◽  
Dna Polymerase ◽  
Dna Helicase ◽  
Dna Lesions ◽  
Replication Forks ◽  
Content Type ◽  
Pol Iv ◽  
Dna Polymerase Iv ◽  
Stalled Replication Forks

ABSTRACTEscherichia coli's DNA polymerase IV (Pol IV/DinB), a member of the Y family of error-prone polymerases, is induced during the SOS response to DNA damage and is responsible for translesion bypass and adaptive (stress-induced) mutation. In this study, the localization of Pol IV after DNA damage was followed using fluorescent fusions. After exposure ofE. colito DNA-damaging agents, fluorescently tagged Pol IV localized to the nucleoid as foci. Stepwise photobleaching indicated ∼60% of the foci consisted of three Pol IV molecules, while ∼40% consisted of six Pol IV molecules. Fluorescently tagged Rep, a replication accessory DNA helicase, was recruited to the Pol IV foci after DNA damage, suggesting that thein vitrointeraction between Rep and Pol IV reported previously also occursin vivo. Fluorescently tagged RecA also formed foci after DNA damage, and Pol IV localized to them. To investigate if Pol IV localizes to double-strand breaks (DSBs), an I-SceI endonuclease-mediated DSB was introduced close to a fluorescently labeled LacO array on the chromosome. After DSB induction, Pol IV localized to the DSB site in ∼70% of SOS-induced cells. RecA also formed foci at the DSB sites, and Pol IV localized to the RecA foci. These results suggest that Pol IV interacts with RecAin vivoand is recruited to sites of DSBs to aid in the restoration of DNA replication.IMPORTANCEDNA polymerase IV (Pol IV/DinB) is an error-prone DNA polymerase capable of bypassing DNA lesions and aiding in the restart of stalled replication forks. In this work, we demonstratein vivolocalization of fluorescently tagged Pol IV to the nucleoid after DNA damage and to DNA double-strand breaks. We show colocalization of Pol IV with two proteins: Rep DNA helicase, which participates in replication, and RecA, which catalyzes recombinational repair of stalled replication forks. Time course experiments suggest that Pol IV recruits Rep and that RecA recruits Pol IV. These findings providein vivoevidence that Pol IV aids in maintaining genomic stability not only by bypassing DNA lesions but also by participating in the restoration of stalled replication forks.

scholarly journals Genotoxicity and Gene Expression in the Rat Lung Tissue following Instillation and Inhalation of Different Variants of Amorphous Silica Nanomaterials (aSiO2 NM)

Nanomaterials ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 1502
Author(s):  
Fátima Brandão ◽  
Carla Costa ◽  
Maria João Bessa ◽  
Elise Dumortier ◽  
Florence Debacq-Chainiaux ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Damage ◽  
Amorphous Silica ◽  
Oxidative Dna Damage ◽  
Intratracheal Instillation ◽  
Dna Lesions ◽  
Lung Cells ◽  
Rat Lung ◽  
Inhalation Study

Several reports on amorphous silica nanomaterial (aSiO2 NM) toxicity have been questioning their safety. Herein, we investigated the in vivo pulmonary toxicity of four variants of aSiO2 NM: SiO2_15_Unmod, SiO2_15_Amino, SiO2_7 and SiO2_40. We focused on alterations in lung DNA and protein integrity, and gene expression following single intratracheal instillation in rats. Additionally, a short-term inhalation study (STIS) was carried out for SiO2_7, using TiO2_NM105 as a benchmark NM. In the instillation study, a significant but slight increase in oxidative DNA damage in rats exposed to the highest instilled dose (0.36 mg/rat) of SiO2_15_Amino was observed in the recovery (R) group. Exposure to SiO2_7 or SiO2_40 markedly increased oxidative DNA lesions in rat lung cells of the exposure (E) group at every tested dose. This damage seems to be repaired, since no changes compared to controls were observed in the R groups. In STIS, a significant increase in DNA strand breaks of the lung cells exposed to 0.5 mg/m3 of SiO2_7 or 50 mg/m3 of TiO2_NM105 was observed in both groups. The detected gene expression changes suggest that oxidative stress and/or inflammation pathways are likely implicated in the induction of (oxidative) DNA damage. Overall, all tested aSiO2 NM were not associated with marked in vivo toxicity following instillation or STIS. The genotoxicity findings for SiO2_7 from instillation and STIS are concordant; however, changes in STIS animals were more permanent/difficult to revert.

scholarly journals Psychomotor stimulant effects of α-pyrrolidinovalerophenone (αPVP) enantiomers correlate with drug binding kinetics at the dopamine transporter

2021 ◽  
Author(s):  
Marco Niello ◽  
Spyridon Sideromenos ◽  
Ralph Gradisch ◽  
Ronan O'Shea ◽  
Jakob Schwazer ◽  
...  
Keyword(s):  
Dopamine Transporter ◽  
In Silico ◽  
Drug Binding ◽  
Binding Kinetics ◽  
Irreversible Binding ◽  
Fast Kinetics ◽  
Slow Dissociation ◽  
Kinetics Of

Abstract α-Pyrrolidinovalerophenone (αPVP) is a psychostimulant and drug of abuse associated with severe intoxications in humans. αPVP exerts long-lasting psychostimulant effects, when compared to the classical dopamine transporter (DAT) inhibitor cocaine. Here, we compared the two enantiomeric forms of αPVP, the R- and the S-αPVP, with cocaine using a combination of in silico, in vitro and in vivo approaches. We found that αPVP enantiomers substantially differ from cocaine in their binding kinetics. The two enantiomers differ from each other in their association rates. However, they show similar slow dissociation rates leading to pseudo-irreversible binding kinetics at DAT. The pseudo-irreversible binding kinetics of αPVP is responsible for the observed non-competitive pharmacology and it correlates with persistent psychostimulant effects in mice. Thus, the slow binding kinetics of αPVP enantiomers profoundly differ from the fast kinetics of cocaine both in vitro and in vivo, suggesting drug-binding kinetics as a potential driver of psychostimulant effects in vivo.

scholarly journals Acquired temozolomide resistance in MGMT-deficient glioblastoma cells is associated with regulation of DNA repair by DHC2

Brain ◽  
2019 ◽  
Vol 142 (8) ◽  
pp. 2352-2366 ◽  
Author(s):  
Guo-zhong Yi ◽  
Guanglong Huang ◽  
Manlan Guo ◽  
Xi’an Zhang ◽  
Hai Wang ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Dna Methyltransferase ◽  
Molecular Mechanisms ◽  
Progression Free Survival ◽  
Recurrent Glioblastoma ◽  
Dna Lesions ◽  
Dna Repair Proteins

Abstract The acquisition of temozolomide resistance is a major clinical challenge for glioblastoma treatment. Chemoresistance in glioblastoma is largely attributed to repair of temozolomide-induced DNA lesions by O6-methylguanine-DNA methyltransferase (MGMT). However, some MGMT-deficient glioblastomas are still resistant to temozolomide, and the underlying molecular mechanisms remain unclear. We found that DYNC2H1 (DHC2) was expressed more in MGMT-deficient recurrent glioblastoma specimens and its expression strongly correlated to poor progression-free survival in MGMT promotor methylated glioblastoma patients. Furthermore, silencing DHC2, both in vitro and in vivo, enhanced temozolomide-induced DNA damage and significantly improved the efficiency of temozolomide treatment in MGMT-deficient glioblastoma. Using a combination of subcellular proteomics and in vitro analyses, we showed that DHC2 was involved in nuclear localization of the DNA repair proteins, namely XPC and CBX5, and knockdown of either XPC or CBX5 resulted in increased temozolomide-induced DNA damage. In summary, we identified the nuclear transportation of DNA repair proteins by DHC2 as a critical regulator of acquired temozolomide resistance in MGMT-deficient glioblastoma. Our study offers novel insights for improving therapeutic management of MGMT-deficient glioblastoma.

Drug-induced DNA damage and tumor chemosensitivity.

1990 ◽  
Vol 8 (12) ◽  
pp. 2062-2084 ◽  
Author(s):  
R J Epstein
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Tumor Cell ◽  
Malignant Disease ◽  
Dna Lesions ◽  
Drug Induced ◽  
Cell Subpopulations ◽  
Therapeutic Cell

Cytotoxic drugs act principally by damaging tumor-cell DNA. Quantitative analysis of this interaction provides a basis for understanding the biology of therapeutic cell kill as well as a rational strategy for optimizing and predicting tumor response. Recent advances have made it possible to correlate assayed DNA lesions with cytotoxicity in tumor cell lines, in animal models, and in patients with malignant disease. In addition, many of the complex interrelationships between DNA damage, DNA repair, and alterations of gene expression in response to DNA damage have been defined. Techniques for modulating DNA damage and cytotoxicity using schedule-specific cytotoxic combinations, DNA repair inhibitors, cell-cycle manipulations, and adjunctive noncytotoxic drug therapy are being developed, and critical therapeutic targets have been identified within tumor-cell subpopulations and genomic DNA alike. Most importantly, methods for predicting clinical response to cytotoxic therapy using both in vitro markers of tumor-cell sensitivity and in vivo measurements of drug-induced DNA damage are now becoming a reality. These advances can be expected to provide a strong foundation for the development of innovative cytotoxic drug strategies over the next decade.

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