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Molecular Cell Jan 2021Replication fork reversal is a global response to replication stress in mammalian cells, but precisely how it occurs remains poorly understood. Here, we show that, upon...
Replication fork reversal is a global response to replication stress in mammalian cells, but precisely how it occurs remains poorly understood. Here, we show that, upon replication stress, DNA topoisomerase IIalpha (TOP2A) is recruited to stalled forks in a manner dependent on the SNF2-family DNA translocases HLTF, ZRANB3, and SMARCAL1. This is accompanied by an increase in TOP2A SUMOylation mediated by the SUMO E3 ligase ZATT and followed by recruitment of a SUMO-targeted DNA translocase, PICH. Disruption of the ZATT-TOP2A-PICH axis results in accumulation of partially reversed forks and enhanced genome instability. These results suggest that fork reversal occurs via a sequential two-step process. First, HLTF, ZRANB3, and SMARCAL1 initiate limited fork reversal, creating superhelical strain in the newly replicated sister chromatids. Second, TOP2A drives extensive fork reversal by resolving the resulting topological barriers and via its role in recruiting PICH to stalled forks.
Topics: DNA Helicases; DNA Replication; DNA Topoisomerases, Type II; DNA-Binding Proteins; Genome, Human; Genomic Instability; HEK293 Cells; HeLa Cells; Humans; Poly-ADP-Ribose Binding Proteins; Transcription Factors
PubMed: 33296677
DOI: 10.1016/j.molcel.2020.11.007 -
International Journal of Molecular... Jul 2023Topoisomerases, common targets for anti-cancer therapeutics, are crucial enzymes for DNA replication, transcription, and many other aspects of DNA metabolism. The... (Review)
Review
Topoisomerases, common targets for anti-cancer therapeutics, are crucial enzymes for DNA replication, transcription, and many other aspects of DNA metabolism. The potential anti-cancer effects of thiosemicarbazones (TSC) and metal-TSC complexes have been demonstrated to target several biological processes, including DNA metabolism. Human topoisomerases were discovered among the molecular targets for TSCs, and metal-chelated TSCs specifically displayed significant inhibition of topoisomerase II. The processes by which metal-TSCs or TSCs inhibit topoisomerases are still being studied. In this brief review, we summarize the TSCs and metal-TSCs that inhibit various types of human topoisomerases, and we note some of the key unanswered questions regarding this interesting class of diverse compounds.
Topics: Humans; Coordination Complexes; DNA Topoisomerases, Type II; Copper; DNA; Thiosemicarbazones; Antineoplastic Agents
PubMed: 37569386
DOI: 10.3390/ijms241512010 -
International Journal of Molecular... Jan 2018Cellular DNA topoisomerases (topo I and topo II) are highly conserved enzymes that regulate the topology of DNA during normal genome transactions, such as DNA... (Review)
Review
Cellular DNA topoisomerases (topo I and topo II) are highly conserved enzymes that regulate the topology of DNA during normal genome transactions, such as DNA transcription and replication. In budding yeast, topo I is dispensable whereas topo II is essential, suggesting fundamental and exclusive roles for topo II, which might include the functions of the topo IIa and topo IIb isoforms found in mammalian cells. In this review, we discuss major findings of the structure and chromosomal organization of genes regulated by topo II in budding yeast. Experimental data was derived from short (10 min) and long term (120 min) responses to topo II inactivation in top-2 ts mutants. First, we discuss how short term responses reveal a subset of yeast genes that are regulated by topo II depending on their promoter architecture. These short term responses also uncovered topo II regulation of transcription across multi-gene clusters, plausibly by common DNA topology management. Finally, we examine the effects of deactivated topo II on the elongation of RNA transcripts. Each study provides an insight into the particular chromatin structure that interacts with the activity of topo II. These findings are of notable clinical interest as numerous anti-cancer therapies interfere with topo II activity.
Topics: Chromatin Assembly and Disassembly; Chromosomes, Fungal; DNA Topoisomerases, Type II; Genes, Fungal; Saccharomyces cerevisiae; Transcriptome
PubMed: 29301361
DOI: 10.3390/ijms19010134 -
International Journal of Molecular... Jan 2023Several reviews of inhibitors of topoisomerase II have been published, covering research before 2018. Therefore, this review is focused primarily on more recent... (Review)
Review
Several reviews of inhibitors of topoisomerase II have been published, covering research before 2018. Therefore, this review is focused primarily on more recent publications with relevant points from the earlier literature. Topoisomerase II is an established target for anticancer drugs, which are further subdivided into poisons and catalytic inhibitors. While most of the topoisomerase II-based drugs in clinical use are mostly topoisomerase II poisons, their mechanism of action has posed severe concern due to DNA damaging potential, including the development of multi-drug resistance. As a result, we are beginning to see a gradual paradigm shift towards non-DNA damaging agents, such as the lesser studied topoisomerase II catalytic inhibitors. In addition, this review describes some novel selective catalytic topoisomerase II inhibitors. The ultimate goal is to bring researchers up to speed by curating and delineating new scaffolds as the leads for the optimization and development of new potent, safe, and selective agents for the treatment of cancer.
Topics: Humans; Topoisomerase II Inhibitors; Antineoplastic Agents; DNA Topoisomerases, Type II; Neoplasms; DNA; Topoisomerase I Inhibitors; Enzyme Inhibitors
PubMed: 36768852
DOI: 10.3390/ijms24032532 -
Nucleic Acids Research Jan 2018Tyrosyl-DNA Phosphodiesterases 1 (TDP1) and 2 (TDP2) are eukaryotic enzymes that clean-up after aberrant topoisomerase activity. While TDP1 hydrolyzes phosphotyrosyl... (Review)
Review
Tyrosyl-DNA Phosphodiesterases 1 (TDP1) and 2 (TDP2) are eukaryotic enzymes that clean-up after aberrant topoisomerase activity. While TDP1 hydrolyzes phosphotyrosyl peptides emanating from trapped topoisomerase I (Top I) from the 3' DNA ends, topoisomerase 2 (Top II)-induced 5'-phosphotyrosyl residues are processed by TDP2. Even though the canonical functions of TDP1 and TDP2 are complementary, they exhibit little structural or sequence similarity. Homozygous mutations in genes encoding these enzymes lead to the development of severe neurodegenerative conditions due to the accumulation of transcription-dependent topoisomerase cleavage complexes underscoring the biological significance of these enzymes in the repair of topoisomerase-DNA lesions in the nervous system. TDP1 can promiscuously process several blocked 3' ends generated by DNA damaging agents and nucleoside analogs in addition to hydrolyzing 3'-phosphotyrosyl residues. In addition, deficiency of these enzymes causes hypersensitivity to anti-tumor topoisomerase poisons. Thus, TDP1 and TDP2 are promising therapeutic targets and their inhibitors are expected to significantly synergize the effects of current anti-tumor therapies including topoisomerase poisons and other DNA damaging agents. This review covers the structural aspects, biology and regulation of these enzymes, along with ongoing developments in the process of discovering safe and effective TDP inhibitors.
Topics: DNA; DNA Damage; DNA Repair; DNA Topoisomerases, Type I; DNA Topoisomerases, Type II; DNA-Binding Proteins; Humans; Neoplasms; Nuclear Proteins; Phosphoric Diester Hydrolases; Protein Binding; Transcription Factors
PubMed: 29216365
DOI: 10.1093/nar/gkx1219 -
Nature May 2022In preparation for mitotic cell division, the nuclear DNA of human cells is compacted into individualized, X-shaped chromosomes. This metamorphosis is driven mainly by...
In preparation for mitotic cell division, the nuclear DNA of human cells is compacted into individualized, X-shaped chromosomes. This metamorphosis is driven mainly by the combined action of condensins and topoisomerase IIα (TOP2A), and has been observed using microscopy for over a century. Nevertheless, very little is known about the structural organization of a mitotic chromosome. Here we introduce a workflow to interrogate the organization of human chromosomes based on optical trapping and manipulation. This allows high-resolution force measurements and fluorescence visualization of native metaphase chromosomes to be conducted under tightly controlled experimental conditions. We have used this method to extensively characterize chromosome mechanics and structure. Notably, we find that under increasing mechanical load, chromosomes exhibit nonlinear stiffening behaviour, distinct from that predicted by classical polymer models. To explain this anomalous stiffening, we introduce a hierarchical worm-like chain model that describes the chromosome as a heterogeneous assembly of nonlinear worm-like chains. Moreover, through inducible degradation of TOP2A specifically in mitosis, we provide evidence that TOP2A has a role in the preservation of chromosome compaction. The methods described here open the door to a wide array of investigations into the structure and dynamics of both normal and disease-associated chromosomes.
Topics: Chromosomes; Chromosomes, Human; DNA; DNA Topoisomerases, Type II; Humans; Mitosis; Optics and Photonics
PubMed: 35508652
DOI: 10.1038/s41586-022-04666-5 -
Viruses Dec 2022Although many arenaviruses cause severe diseases with high fatality rates each year, treatment options are limited to off-label use of ribavirin, and a Food and Drug...
Although many arenaviruses cause severe diseases with high fatality rates each year, treatment options are limited to off-label use of ribavirin, and a Food and Drug Administration (FDA)-approved vaccine is not available. To identify novel therapeutic candidates against arenaviral diseases, an RNA polymerase I-driven minigenome (MG) expression system for Lassa virus (LASV) was developed and optimized for high-throughput screening (HTS). Using this system, we screened 2595 FDA-approved compounds for inhibitors of LASV genome replication and identified multiple compounds including pixantrone maleate, a topoisomerase II inhibitor, as hits. Other tested topoisomerase II inhibitors also suppressed LASV MG activity. These topoisomerase II inhibitors also inhibited Junin virus (JUNV) MG activity and effectively limited infection by the JUNV Candid #1 strain, and siRNA knockdown of both topoisomerases (IIα and IIβ) restricted JUNV replication. These results suggest that topoisomerases II regulate arenavirus replication and can serve as molecular targets for panarenaviral replication inhibitors.
Topics: Antiviral Agents; Arenavirus; DNA Topoisomerases, Type II; Junin virus; Lassa virus; Topoisomerase II Inhibitors; Humans
PubMed: 36680145
DOI: 10.3390/v15010105 -
The Journal of Cell Biology Sep 2017The size of mitotic chromosomes is coordinated with cell size in a manner dependent on nuclear trafficking. In this study, we conducted an RNA interference screen of the...
The size of mitotic chromosomes is coordinated with cell size in a manner dependent on nuclear trafficking. In this study, we conducted an RNA interference screen of the nucleome in a strain carrying an exceptionally long chromosome and identified the centromere-specific histone H3 variant CENP-A and the DNA decatenizing enzyme topoisomerase-II (topo-II) as candidate modulators of chromosome size. In the holocentric organism , CENP-A is positioned periodically along the entire length of chromosomes, and in mitosis, these genomic regions come together linearly to form the base of kinetochores. We show that CENP-A protein levels decreased through development coinciding with chromosome-size scaling. Partial loss of CENP-A protein resulted in shorter mitotic chromosomes, consistent with a role in setting chromosome length. Conversely, topo-II levels were unchanged through early development, and partial topo-II depletion led to longer chromosomes. Topo-II localized to the perimeter of mitotic chromosomes, excluded from the centromere regions, and depletion of topo-II did not change CENP-A levels. We propose that self-assembly of centromeric chromatin into an extended linear array promotes elongation of the chromosome, whereas topo-II promotes chromosome-length shortening.
Topics: Animals; Autoantigens; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Centromere Protein A; Chromatin; Chromatin Assembly and Disassembly; Chromosomal Proteins, Non-Histone; Chromosomes; DNA Topoisomerases, Type II; Gene Expression Regulation, Developmental; Kinetochores; Mitosis; RNA Interference
PubMed: 28733327
DOI: 10.1083/jcb.201608084 -
MBio Oct 2023African swine fever virus (ASFV) is a highly contagious virus that causes lethal hemorrhagic diseases known as African swine fever (ASF) with a case fatality rate of...
African swine fever virus (ASFV) is a highly contagious virus that causes lethal hemorrhagic diseases known as African swine fever (ASF) with a case fatality rate of 100%. There is an urgent need to develop anti-ASFV drugs. We determine the first high-resolution structures of viral topoisomerase ASFV P1192R in both the closed and open C-gate forms. P1192R shows a similar overall architecture with eukaryotic and prokaryotic type II topoisomerases, which have been successful targets of many antimicrobials and anticancer drugs, with the most similarity to yeast topo II. P1192R also exhibits differences in the details of active site configuration, which are important to enzyme activity. These two structures offer useful structural information for antiviral drug design and provide structural evidence to support that eukaryotic type IIA topoisomerase likely originated from horizontal gene transfer from the virus.
Topics: Swine; Animals; African Swine Fever Virus; African Swine Fever; Cryoelectron Microscopy; DNA Topoisomerases, Type II; Catalytic Domain; Saccharomyces cerevisiae
PubMed: 37610250
DOI: 10.1128/mbio.01228-23 -
Molecular Biology Reports Sep 2021DNA topoisomerases II (TOP2) are peculiar enzymes (TOP2α and TOP2β) that modulate the conformation of DNA by momentarily breaking double-stranded DNA to allow another... (Review)
Review
DNA topoisomerases II (TOP2) are peculiar enzymes (TOP2α and TOP2β) that modulate the conformation of DNA by momentarily breaking double-stranded DNA to allow another strand to pass through, and then rejoins the DNA phosphodiester backbone. TOP2α and TOP2β play vital roles in nearly all events involving DNA metabolism, including DNA transcription, replication, repair, and chromatin remodeling. Beyond these vital functions, TOP2 enzymes are therapeutic targets for various anticancer drugs, termed TOP2 poisons, such as teniposide, etoposide, and doxorubicin. These drugs exert their antitumor activity by inhibiting the activity of TOP2-DNA cleavage complexes (TOP2ccs) containing DNA double-strand breaks (DSBs), subsequently leading to the degradation of TOP2 by the 26S proteasome, thereby exposing the DSBs and eliciting a DNA damage response. Failure of the DSBs to be appropriately repaired leads to genomic instability. Due to this mechanism, patients treated with TOP2-based drugs have a high incidence of secondary malignancies and cardiotoxicity. While the cytotoxicity associated with TOP2 poisons appears to be TOP2α-dependent, the DNA sequence rearrangements and formation of DSBs appear to be mediated primarily through TOP2β inhibition, likely due to the differential degradation patterns of TOP2α and TOP2β. Research over the past few decades has shown that under various conditions, the ubiquitin-proteasome system (UPS) and the SUMOylation pathway are primarily responsible for regulating the stability and activity of TOP2 and are therefore critical regulators of the therapeutic effect of TOP2-targeting drugs. In this review, we summarize the current progress on the regulation of TOP2α and TOP2β by ubiquitination and SUMOylation. By fully elucidating the basic biology of these essential and complex molecular mechanisms, better strategies may be developed to improve the therapeutic efficacy of TOP2 poisons and minimize the risks of therapy-related secondary malignancy.
Topics: Antineoplastic Agents; Cardiotoxicity; DNA Breaks, Double-Stranded; DNA Topoisomerases, Type II; Humans; Neoplasms; Poly-ADP-Ribose Binding Proteins; Proteasome Endopeptidase Complex; Sumoylation; Topoisomerase II Inhibitors; Treatment Outcome
PubMed: 34476738
DOI: 10.1007/s11033-021-06665-7