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Current Genetics Dec 2019We use genetic assays to suggest that transcription-coupled repair or new origin formation in Escherichia coli involves removal of RNAP to create an RNA primer for DNA... (Review)
Review
We use genetic assays to suggest that transcription-coupled repair or new origin formation in Escherichia coli involves removal of RNAP to create an RNA primer for DNA synthesis. Transcription factor DksA was shown to play a role in numerous reactions involving RNA polymerase. Some, but not all, of the activities of DksA at promoters or during transcription elongation require (p)ppGpp. In addition to its role during transcription, DksA is also involved in maintaining genome integrity. Cells lacking DksA are sensitive to multiple DNA damaging agents including UV light, ionizing radiation, mitomycin C, and nalidixic acid. Here, we focus on two recent studies addressing the importance of DksA in the repair of double-strand breaks (DSBs), one by Sivaramakrishnan et al. (Nature 550:214-218, 2017) and one originating in our laboratory, Myka et al. (Mol Microbiol 111:1382-1397. https://doi.org/10.1111/mmi.14227 , 2019). It appears that depending on the type and possibly location of DNA damage, DksA can play either a passive or an active role in DSB repair. The passive role relies on exclusion of anti-backtracking factors from the RNAP secondary channel. The exact mechanism of active DksA-mediated DNA repair is unknown. However, DksA was proposed to destabilize transcription complexes, thus clearing the way for recombination and DNA repair. Based on the requirement for DksA, both in repair of DSBs and the R-loop-dependent formation of new origins of DNA replication, we propose that DksA may allow for removal of RNAP without unwinding of the RNA:DNA hybrid, which can then be extended by a DNA polymerase. This mechanism obviates the need for RNAP backtracking to repair damaged DNA.
Topics: DNA Breaks, Double-Stranded; DNA Repair; DNA Topoisomerases, Type II; DNA-Directed RNA Polymerases; Escherichia coli; Escherichia coli Proteins; Guanosine Pentaphosphate; Nalidixic Acid; Phleomycins; Promoter Regions, Genetic; Transcription Factors
PubMed: 31076845
DOI: 10.1007/s00294-019-00983-x -
Science Advances Dec 2023The catalytic cycle of topoisomerase 2 (TOP2) enzymes proceeds via a transient DNA double-strand break (DSB) intermediate termed the TOP2 cleavage complex (TOP2cc), in...
The catalytic cycle of topoisomerase 2 (TOP2) enzymes proceeds via a transient DNA double-strand break (DSB) intermediate termed the TOP2 cleavage complex (TOP2cc), in which the TOP2 protein is covalently bound to DNA. Anticancer agents such as etoposide operate by stabilizing TOP2ccs, ultimately generating genotoxic TOP2-DNA protein cross-links that require processing and repair. Here, we identify RAD54 like 2 (RAD54L2) as a factor promoting TOP2cc resolution. We demonstrate that RAD54L2 acts through a novel mechanism together with zinc finger protein associated with tyrosyl-DNA phosphodiesterase 2 (TDP2) and TOP2 (ZATT/ZNF451) and independent of TDP2. Our work suggests a model wherein RAD54L2 recognizes sumoylated TOP2 and, using its ATPase activity, promotes TOP2cc resolution and prevents DSB exposure. These findings suggest RAD54L2-mediated TOP2cc resolution as a potential mechanism for cancer therapy resistance and highlight RAD54L2 as an attractive candidate for drug discovery.
Topics: Humans; DNA Adducts; DNA-Binding Proteins; Phosphoric Diester Hydrolases; DNA Topoisomerases, Type II; DNA; Genomic Instability; DNA Helicases
PubMed: 38055822
DOI: 10.1126/sciadv.adl2108 -
Molecules (Basel, Switzerland) Mar 2021A cannabinoid anticancer para-quinone, HU-331, which was synthesized by our group five decades ago, was shown to have very high efficacy against human cancer cell lines... (Review)
Review
A cannabinoid anticancer para-quinone, HU-331, which was synthesized by our group five decades ago, was shown to have very high efficacy against human cancer cell lines in-vitro and against in-vivo grafts of human tumors in nude mice. The main mechanism was topoisomerase IIα catalytic inhibition. Later, several groups synthesized related compounds. In the present presentation, we review the publications on compounds synthesized on the basis of HU-331, summarize their published activities and mechanisms of action and report the synthesis and action of novel quinones, thus expanding the structure-activity relationship in these series.
Topics: Animals; Cannabidiol; DNA Topoisomerases, Type II; Humans; Mice; Mice, Nude; Neoplasm Proteins; Neoplasms, Experimental; Poly-ADP-Ribose Binding Proteins; Quinones; Topoisomerase II Inhibitors
PubMed: 33801057
DOI: 10.3390/molecules26061761 -
Nature Chemical Biology May 2023Etoposide is a broadly employed chemotherapeutic and eukaryotic topoisomerase II poison that stabilizes cleaved DNA intermediates to promote DNA breakage and...
Etoposide is a broadly employed chemotherapeutic and eukaryotic topoisomerase II poison that stabilizes cleaved DNA intermediates to promote DNA breakage and cytotoxicity. How etoposide perturbs topoisomerase dynamics is not known. Here we investigated the action of etoposide on yeast topoisomerase II, human topoisomerase IIα and human topoisomerase IIβ using several sensitive single-molecule detection methods. Unexpectedly, we found that etoposide induces topoisomerase to trap DNA loops, compacting DNA and restructuring DNA topology. Loop trapping occurs after ATP hydrolysis but before strand ejection from the enzyme. Although etoposide decreases the innate stability of topoisomerase dimers, it increases the ability of the enzyme to act as a stable roadblock. Interestingly, the three topoisomerases show similar etoposide-mediated resistance to dimer separation and sliding along DNA but different abilities to compact DNA and chirally relax DNA supercoils. These data provide unique mechanistic insights into the functional consequences of etoposide on topoisomerase II dynamics.
Topics: Humans; Etoposide; Topoisomerase II Inhibitors; DNA Topoisomerases, Type II; DNA
PubMed: 36717711
DOI: 10.1038/s41589-022-01235-9 -
The Prostate Dec 2023Castration-resistant prostate cancer (CRPC) is refractory to hormone treatment and the therapeutic options are continuously advancing. This study aims to discover the...
BACKGROUND
Castration-resistant prostate cancer (CRPC) is refractory to hormone treatment and the therapeutic options are continuously advancing. This study aims to discover the anti-CRPC effects and underlying mechanisms of small-molecule compounds targeting topoisomerase (TOP) II and cellular components of DNA damage repair.
METHODS
Cell proliferation was determined in CRPC PC-3 and DU-145 cells using anchorage-dependent colony formation, sulforhodamine B assay and flow cytometric analysis of CFSE staining. Flow cytometric analyses of propidium iodide staining and JC-1 staining were used to examine the population of cell-cycle phases and mitochondrial membrane potential, respectively. Nuclear extraction was performed to detect the nuclear localization of cellular components in DNA repair pathways. Protein expressions were determined using Western blot analysis.
RESULTS
A series of azathioxanthone-based derivatives were synthesized and examined for bioactivities in which WC-A13, WC-A14, WC-A15, and WC-A16 displayed potent anti-CRPC activities in both PC-3 and DU-145 cell models. These WC-A compounds selectively downregulated both TOP IIα and TOP IIβ but not TOP I protein expression. WC-A13, WC-A14, and WC-A15 were more potent than WC-A16 on TOP II inhibition, mitochondrial dysfunction, and induction of caspase cascades indicating the key role of amine-containing side chain of the compounds in determining anti-CRPC activities. Furthermore, WC-A compounds induced an increase of γH2AX and activated ATR-Chk1 and ATM-Chk2 signaling pathways. P21 protein expression was also upregulated by WC-A compounds in which WC-A16 showed the least activity. Notably, WC-A compounds exhibited different regulation on Rad51, a major protein in homologous recombination of DNA in double-stranded break repair. WC-A13, WC-A14, and WC-A15 inhibited, whereas WC-A16 induced, the nuclear translocation of Rad51.
CONCLUSION
The data suggest that WC-A compounds exhibit anti-CRPC effects through the inhibition of TOP II activities, leading to mitochondrial stress-involved caspase activation and apoptosis. Moreover, WC-A13, WC-A14, and WC-A15 but not WC-A16 display inhibitory activities of Rad51-mediated DNA repair pathway which may increase apoptotic effect of CRPC cells.
Topics: Male; Humans; Antineoplastic Agents; Prostatic Neoplasms, Castration-Resistant; Cell Line, Tumor; Apoptosis; Cell Proliferation; Caspases; DNA Repair; DNA Topoisomerases, Type II
PubMed: 37583103
DOI: 10.1002/pros.24613 -
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 -
Biochemistry Jun 2021The extensive length, compaction, and interwound nature of DNA, together with its controlled and restricted movement in eukaryotic cells, create a number of topological... (Review)
Review
The extensive length, compaction, and interwound nature of DNA, together with its controlled and restricted movement in eukaryotic cells, create a number of topological issues that profoundly affect all of the functions of the genetic material. Topoisomerases are essential enzymes that modulate the topological structure of the double helix, including the regulation of DNA under- and overwinding and the removal of tangles and knots from the genome. Type II topoisomerases alter DNA topology by generating a transient double-stranded break in one DNA segment and allowing another segment to pass through the DNA gate. These enzymes are involved in a number of critical nuclear processes in eukaryotic cells, such as DNA replication, transcription, and recombination, and are required for proper chromosome structure and segregation. However, because type II topoisomerases generate double-stranded breaks in the genetic material, they also are intrinsically dangerous enzymes that have the capacity to fragment the genome. As a result of this dualistic nature, type II topoisomerases are the targets for a number of widely prescribed anticancer drugs. This article will describe the structure and catalytic mechanism of eukaryotic type II topoisomerases and will go on to discuss the actions of topoisomerase II poisons, which are compounds that stabilize DNA breaks generated by the type II enzyme and convert these essential enzymes into "molecular scissors." Topoisomerase II poisons represent a broad range of structural classes and include anticancer drugs, dietary components, and environmental chemicals.
Topics: Antineoplastic Agents; DNA; DNA Damage; DNA Topoisomerases, Type II; Eukaryota; Genome; Humans; Topoisomerase II Inhibitors; Translocation, Genetic
PubMed: 34008964
DOI: 10.1021/acs.biochem.1c00240 -
Bioorganic & Medicinal Chemistry Aug 2023Topoisomerases are key molecular enzymes responsible for altering DNA topology, thus they have long been considered as attractive targets for novel chemotherapeutic...
Topoisomerases are key molecular enzymes responsible for altering DNA topology, thus they have long been considered as attractive targets for novel chemotherapeutic agents. Topoisomerase type II (Topo II) catalytic inhibitors embrace a fresh perspective meant to get beyond drawbacks caused by topo II poisons, such as cardiotoxicity and secondary malignancies. Based on previously reported 5H-indeno[1,2-b]pyridines, here we presented new twenty-three hybrid di-indenopyridines along with their topo I/IIα inhibitory and antiproliferative activity. Most of the prepared 11-phenyl-diindenopyridines showed negligible topo I inhibitory activity, showing selectivity over topo II. Among the series, we finally selected compound 17, which displayed 100 % topo IIα inhibition at 20 μM concentration and comparable antiproliferative activity against the tested cell lines. Through competitive EtBr displacement assay, cleavable complex assay, and comet assay, compound 17 was finally determined as a non-intercalative catalytic topo IIα inhibitor. The findings in this study highlight the significance of phenolic, halophenyl, thienyl, and furyl groups at the 4-position of the indane ring in the design and synthesis of di-indenopyridines as potent catalytic topo IIα inhibitors with remarkable anticancer effects.
Topics: Cell Line, Tumor; Structure-Activity Relationship; Antineoplastic Agents; Topoisomerase II Inhibitors; DNA Topoisomerases, Type II; Cell Proliferation
PubMed: 37418826
DOI: 10.1016/j.bmc.2023.117403 -
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