Suppressing proteasome mediated processing of topoisomerase II DNA-protein complexes preserves genome integrity

Topoisomerase II (TOP2) relieves topological stress in DNA by introducing double-strand breaks (DSBs) via a transient, covalently linked TOP2 DNA-protein intermediate, termed TOP2 cleavage complex (TOP2cc). TOP2ccs are normally rapidly reversible, but can be stabilized by TOP2 poisons, such as the chemotherapeutic agent etoposide (ETO). TOP2 poisons have shown significant variability in their therapeutic effectiveness across different cancers for reasons that remain to be determined. One potential explanation for the differential cellular response to these drugs is in the manner by which cells process TOP2ccs. Cells are thought to remove TOP2ccs primarily by proteolytic degradation followed by DNA DSB repair. Here, we show that proteasome-mediated repair of TOP2cc is highly error-prone. Pre-treating primary splenic mouse B-cells with proteasome inhibitors prevented the proteolytic processing of trapped TOP2ccs, suppressed the DNA damage response (DDR) and completely protected cells from ETO-induced genome instability, thereby preserving cellular viability. When degradation of TOP2cc was suppressed, the TOP2 enzyme uncoupled itself from the DNA following ETO washout, in an error-free manner. This suggests a potential mechanism of developing resistance to topoisomerase poisons by ensuring rapid TOP2cc reversal.

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Four-pronged negative feedback of DSB machinery in meiotic DNA-break control in mice

Nucleic Acids Research ◽

10.1093/nar/gkab082 ◽

2021 ◽

Author(s):

Ihsan Dereli ◽

Marcello Stanzione ◽

Fabrizio Olmeda ◽

Frantzeskos Papanikos ◽

Marek Baumann ◽

...

Keyword(s):

Negative Feedback ◽

Protein Complexes ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Homologous Chromosomes ◽

Damage Response ◽

Auxiliary Protein ◽

Genomic Locations ◽

Spatiotemporal Control

Abstract In most taxa, halving of chromosome numbers during meiosis requires that homologous chromosomes (homologues) pair and form crossovers. Crossovers emerge from the recombination-mediated repair of programmed DNA double-strand breaks (DSBs). DSBs are generated by SPO11, whose activity requires auxiliary protein complexes, called pre-DSB recombinosomes. To elucidate the spatiotemporal control of the DSB machinery, we focused on an essential SPO11 auxiliary protein, IHO1, which serves as the main anchor for pre-DSB recombinosomes on chromosome cores, called axes. We discovered that DSBs restrict the DSB machinery by at least four distinct pathways in mice. Firstly, by activating the DNA damage response (DDR) kinase ATM, DSBs restrict pre-DSB recombinosome numbers without affecting IHO1. Secondly, in their vicinity, DSBs trigger IHO1 depletion mainly by another DDR kinase, ATR. Thirdly, DSBs enable homologue synapsis, which promotes the depletion of IHO1 and pre-DSB recombinosomes from synapsed axes. Finally, DSBs and three DDR kinases, ATM, ATR and PRKDC, enable stage-specific depletion of IHO1 from all axes. We hypothesize that these four negative feedback pathways protect genome integrity by ensuring that DSBs form without excess, are well-distributed, and are restricted to genomic locations and prophase stages where DSBs are functional for promoting homologue pairing and crossover formation.

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Bioflavonoids cause DNA double-strand breaks and chromosomal translocations through topoisomerase II-dependent and -independent mechanisms

Mutation Research/Genetic Toxicology and Environmental Mutagenesis ◽

10.1016/j.mrgentox.2020.503144 ◽

2020 ◽

Vol 849 ◽

pp. 503144 ◽

Cited By ~ 1

Author(s):

Donna Goodenow ◽

Faith Emmanuel ◽

Chase Berman ◽

Mark Sahyouni ◽

Christine Richardson

Keyword(s):

Topoisomerase Ii ◽

Double Strand Breaks ◽

Chromosomal Translocations ◽

Dna Double Strand Breaks ◽

Strand Breaks

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Live-cell chromosome dynamics in Arabidopsis thaliana reveals increased chromatin mobility in response to DNA damage

10.1101/2021.11.03.466744 ◽

2021 ◽

Author(s):

Anis Meschichi ◽

Adrien Sicard ◽

Frédéric Pontvianne ◽

Svenja Reeck ◽

Stefanie Rosa

Keyword(s):

Dna Damage ◽

Arabidopsis Thaliana ◽

Genome Instability ◽

High Mobility ◽

Double Strand Breaks ◽

Nuclear Space ◽

Strand Breaks ◽

Homologous Sequences ◽

Damage Response ◽

Repair Mechanisms

Double-strand breaks (DSBs) are a particularly deleterious type of DNA damage potentially leading to translocations and genome instability. Homologous recombination (HR) is a conservative repair pathway in which intact homologous sequences are used as a template for repair. How damaged DNA molecules search for homologous sequences in the crowded space of the cell nucleus is, however, still poorly understood, especially in plants. Here, we measured global chromosome and DSB site mobility, in Arabidopsis thaliana, by tracking the motion of specific loci using the lacO/LacI tagging system and two GFP-tagged HR regulators, RAD51 and RAD54. We observed an increase in chromatin mobility upon the induction of DNA damage, specifically at the S/G2 phases of the cell cycle. Importantly, this increase in mobility was lost on sog1-1 mutant, a central transcription factor of the DNA damage response (DDR), indicating that repair mechanisms actively regulate chromatin mobility upon DNA damage. Interestingly, we observed that DSB sites show remarkably high mobility levels at the early HR stage. Subsequently, a drastic decrease of DSB mobility is observed, which seems to be associated to the relocation of DSBs to the nucleus periphery. Altogether, our data suggest that changes in chromatin mobility are triggered in response to DNA damage, and that this may act as a mechanism to enhance the physical search within the nuclear space to locate a homologous template during homology-directed DNA repair.

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The regulation of the DNA damage response at telomeres: focus on kinases

Biochemical Society Transactions ◽

10.1042/bst20200856 ◽

2021 ◽

Author(s):

Michela Galli ◽

Chiara Frigerio ◽

Maria Pia Longhese ◽

Michela Clerici

Keyword(s):

Cell Cycle ◽

Cellular Response ◽

Cell Cycle Progression ◽

Binding Proteins ◽

Replicative Senescence ◽

Genome Instability ◽

Cycle Progression ◽

Strand Breaks ◽

Cycle Arrest ◽

Linear Chromosomes

The natural ends of linear chromosomes resemble those of accidental double-strand breaks (DSBs). DSBs induce a multifaceted cellular response that promotes the repair of lesions and slows down cell cycle progression. This response is not elicited at chromosome ends, which are organized in nucleoprotein structures called telomeres. Besides counteracting DSB response through specialized telomere-binding proteins, telomeres also prevent chromosome shortening. Despite of the different fate of telomeres and DSBs, many proteins involved in the DSB response also localize at telomeres and participate in telomere homeostasis. In particular, the DSB master regulators Tel1/ATM and Mec1/ATR contribute to telomere length maintenance and arrest cell cycle progression when chromosome ends shorten, thus promoting a tumor-suppressive process known as replicative senescence. During senescence, the actions of both these apical kinases and telomere-binding proteins allow checkpoint activation while bulk DNA repair activities at telomeres are still inhibited. Checkpoint-mediated cell cycle arrest also prevents further telomere erosion and deprotection that would favor chromosome rearrangements, which are known to increase cancer-associated genome instability. This review summarizes recent insights into functions and regulation of Tel1/ATM and Mec1/ATR at telomeres both in the presence and in the absence of telomerase, focusing mainly on discoveries in budding yeast.

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The ATM protein: The importance of being active

The Journal of Cell Biology ◽

10.1083/jcb.201207063 ◽

2012 ◽

Vol 198 (3) ◽

pp. 273-275 ◽

Cited By ~ 11

Author(s):

Yosef Shiloh ◽

Yael Ziv

Keyword(s):

Cellular Response ◽

Cancer Therapeutics ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Ataxia Telangiectasia Mutated ◽

Strand Breaks ◽

Cell Biol ◽

Response Network ◽

Damage Response ◽

Atm Protein

The ataxia telangiectasia mutated (ATM) protein kinase regulates the cellular response to deoxyribonucleic acid (DNA) double-strand breaks by phosphorylating numerous players in the extensive DNA damage response network. Two papers in this issue (Daniel et al. 2012. J. Cell Biol. http://dx.doi.org/10.1083/jcb201204035; Yamamoto et al. 2012. J. Cell Biol. http://dx.doi.org/10.1083/jcb201204098) strikingly show that, in mice, the presence of a catalytically inactive version of ATM is embryonically lethal. This is surprising because mice completely lacking ATM have a much more moderate phenotype. The findings impact on basic cancer research and cancer therapeutics.

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Surveillance of Different Recombination Defects in Mouse Spermatocytes Yields Distinct Responses despite Elimination at an Identical Developmental Stage

Molecular and Cellular Biology ◽

10.1128/mcb.25.16.7203-7215.2005 ◽

2005 ◽

Vol 25 (16) ◽

pp. 7203-7215 ◽

Cited By ~ 137

Author(s):

Marco Barchi ◽

Shantha Mahadevaiah ◽

Monica Di Giacomo ◽

Frédéric Baudat ◽

Dirk G. de Rooij ◽

...

Keyword(s):

Cellular Response ◽

Stage Iv ◽

Development Stage ◽

Male Meiosis ◽

Double Strand Breaks ◽

Dsb Repair ◽

Strand Breaks ◽

Epistasis Analysis ◽

Associated Proteins ◽

Normal Males

ABSTRACT Fundamentally different recombination defects cause apoptosis of mouse spermatocytes at the same stage in development, stage IV of the seminiferous epithelium cycle, equivalent to mid-pachynema in normal males. To understand the cellular response(s) that triggers apoptosis, we examined markers of spermatocyte development in mice with different recombination defects. In Spo11 − / − mutants, which lack the double-strand breaks (DSBs) that initiate recombination, spermatocytes express markers of early to mid-pachynema, forming chromatin domains that contain sex body-associated proteins but that rarely encompass the sex chromosomes. Dmc1 − / − spermatocytes, impaired in DSB repair, appear to arrest at or about late zygonema. Epistasis analysis reveals that this earlier arrest is a response to unrepaired DSBs, and cytological analysis implicates the BRCT-containing checkpoint protein TOPBP1. Atm − / − spermatocytes show similarities to Dmc1 − / − spermatocytes, suggesting that ATM promotes meiotic DSB repair. Msh5 − / − mutants display a set of characteristics distinct from these other mutants. Thus, despite equivalent stages of spermatocyte elimination, different recombination-defective mutants manifest distinct responses, providing insight into surveillance mechanisms in male meiosis.

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