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1
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Amoiridis M, Verigos J, Meaburn K, Gittens WH, Ye T, Neale MJ, Soutoglou E. Inhibition of topoisomerase 2 catalytic activity impacts the integrity of heterochromatin and repetitive DNA and leads to interlinks between clustered repeats. Nat Commun 2024; 15:5727. [PMID: 38977669 PMCID: PMC11231352 DOI: 10.1038/s41467-024-49816-7] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/03/2023] [Accepted: 06/14/2024] [Indexed: 07/10/2024] Open
Abstract
DNA replication and transcription generate DNA supercoiling, which can cause topological stress and intertwining of daughter chromatin fibers, posing challenges to the completion of DNA replication and chromosome segregation. Type II topoisomerases (Top2s) are enzymes that relieve DNA supercoiling and decatenate braided sister chromatids. How Top2 complexes deal with the topological challenges in different chromatin contexts, and whether all chromosomal contexts are subjected equally to torsional stress and require Top2 activity is unknown. Here we show that catalytic inhibition of the Top2 complex in interphase has a profound effect on the stability of heterochromatin and repetitive DNA elements. Mechanistically, we find that catalytically inactive Top2 is trapped around heterochromatin leading to DNA breaks and unresolved catenates, which necessitate the recruitment of the structure specific endonuclease, Ercc1-XPF, in an SLX4- and SUMO-dependent manner. Our data are consistent with a model in which Top2 complex resolves not only catenates between sister chromatids but also inter-chromosomal catenates between clustered repetitive elements.
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Affiliation(s)
- Michalis Amoiridis
- Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Brighton, BN1 9RH, UK
- Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Illkirch, France
- Institut National de la Santé et de la Recherche Médicale (INSERM), U1258, Illkirch, France
- Centre National de Recherche Scientifique (CNRS), UMR7104, Illkirch, France
- Université de Strasbourg, Illkirch, France
| | - John Verigos
- Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Brighton, BN1 9RH, UK
| | - Karen Meaburn
- Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Brighton, BN1 9RH, UK
| | - William H Gittens
- Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Brighton, BN1 9RH, UK
| | - Tao Ye
- Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Illkirch, France
- Institut National de la Santé et de la Recherche Médicale (INSERM), U1258, Illkirch, France
- Centre National de Recherche Scientifique (CNRS), UMR7104, Illkirch, France
- Université de Strasbourg, Illkirch, France
| | - Matthew J Neale
- Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Brighton, BN1 9RH, UK
| | - Evi Soutoglou
- Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Brighton, BN1 9RH, UK.
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2
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Shu J, Jiang J, Wang X, Yang X, Zhao G, Cai T. MDM2 provides TOP2 poison resistance by promoting proteolysis of TOP2βcc in a p53-independent manner. Cell Death Dis 2024; 15:83. [PMID: 38263255 PMCID: PMC10806188 DOI: 10.1038/s41419-024-06474-3] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/15/2023] [Revised: 01/14/2024] [Accepted: 01/15/2024] [Indexed: 01/25/2024]
Abstract
DNA topoisomerase II (TOP2) is an enzyme that performs a critical function in manipulating DNA topology during replication, transcription, and chromosomal compaction by forming a vital intermediate known as the TOP2-DNA cleavage complex (TOP2cc). Although the TOP2cc is often transient, stabilization can be achieved by TOP2 poisons, a family of anti-cancer chemotherapeutic agents targeting TOP2, such as etoposide (VP-16), and then induce double-strand breaks (DSBs) in cellular DNA. TOP2cc first needs to be proteolyzed before it can be processed by TDP2 for the removal of these protein adducts and to produce clean DNA ends necessary for proper repair. However, the mechanism by which TOP2βcc is proteolyzed has not been thoroughly studied. In this study, we report that after exposure to VP-16, MDM2, a RING-type E3 ubiquitin ligase, attaches to TOP2β and initiates polyubiquitination and proteasomal degradation. Mechanistically, during exposure to VP-16, TOP2β binds to DNA to form TOP2βcc, which promotes MDM2 binding and subsequent TOP2β ubiquitination and degradation, and results in a decrease in TOP2βcc levels. Biologically, MDM2 inactivation abrogates TOP2β degradation, stabilizes TOP2βcc, and subsequently increases the number of TOP2β-concealed DSBs, resulting in the rapid death of cancer cells via the apoptotic process. Furthermore, we demonstrate the combination activity of VP-16 and RG7112, an MDM2 inhibitor, in the xenograft tumor model and in situ lung cancer mouse model. Taken together, the results of our research reveal an underlying mechanism by which MDM2 promotes cancer cell survival in the presence of TOP2 poisons by activating proteolysis of TOP2βcc in a p53-independent manner, and provides a rationale for the combination of MDM2 inhibitors with TOP2 poisons for cancer therapy.
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Affiliation(s)
- Jianfeng Shu
- Department of Thoracic Surgery, Ningbo No.2 Hospital, Ningbo, 315010, Zhejiang, China
- Ningbo Institute of Life and Health Industry, University of Chinese Academy of Sciences, Ningbo, 315000, Zhejiang, China
| | - Jinni Jiang
- Department of Thoracic Surgery, Ningbo No.2 Hospital, Ningbo, 315010, Zhejiang, China
- Ningbo Institute of Life and Health Industry, University of Chinese Academy of Sciences, Ningbo, 315000, Zhejiang, China
| | - Xiaofang Wang
- Ningbo Institute of Life and Health Industry, University of Chinese Academy of Sciences, Ningbo, 315000, Zhejiang, China
| | - Xuejie Yang
- Ningbo Institute of Life and Health Industry, University of Chinese Academy of Sciences, Ningbo, 315000, Zhejiang, China
| | - Guofang Zhao
- Department of Thoracic Surgery, Ningbo No.2 Hospital, Ningbo, 315010, Zhejiang, China.
| | - Ting Cai
- Department of Thoracic Surgery, Ningbo No.2 Hospital, Ningbo, 315010, Zhejiang, China.
- Ningbo Institute of Life and Health Industry, University of Chinese Academy of Sciences, Ningbo, 315000, Zhejiang, China.
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3
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Neha S, Dholaniya PS. The Prevailing Role of Topoisomerase 2 Beta and its Associated Genes in Neurons. Mol Neurobiol 2021; 58:6443-6459. [PMID: 34546528 DOI: 10.1007/s12035-021-02561-0] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/12/2021] [Accepted: 09/11/2021] [Indexed: 12/01/2022]
Abstract
Topoisomerase 2 beta (TOP2β) is an enzyme that alters the topological states of DNA by making a transient double-strand break during the transcription process. The direct interaction of TOP2β with DNA strand results in transcriptional regulation of certain genes and some studies have suggested that a particular set of genes are regulated by TOP2β, which have a prominent role in various stages of neuron from development to degeneration. In this review, we discuss the role of TOP2β in various phases of the neuron's life. Based on the existing reports, we have compiled the list of genes, which are directly regulated by the enzyme, from different studies and performed their functional classification. We discuss the role of these genes in neurogenesis, neuron migration, fate determination, differentiation and maturation, generation of neural circuits, and senescence.
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Affiliation(s)
- Neha S
- Department of Biotechnology and Bioinformatics, School of Life Sciences, University of Hyderabad, Hyderabad, Telangana, 500 046, India
| | - Pankaj Singh Dholaniya
- Department of Biotechnology and Bioinformatics, School of Life Sciences, University of Hyderabad, Hyderabad, Telangana, 500 046, India.
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4
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Singh BN, Achary VMM, Panditi V, Sopory SK, Reddy MK. Dynamics of tobacco DNA topoisomerases II in cell cycle regulation: to manage topological constrains during replication, transcription and mitotic chromosome condensation and segregation. PLANT MOLECULAR BIOLOGY 2017; 94:595-607. [PMID: 28634865 DOI: 10.1007/s11103-017-0626-4] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/22/2016] [Accepted: 06/13/2017] [Indexed: 05/21/2023]
Abstract
KEY MESSAGE The topoisomerase II expression varies as a function of cell proliferation. Maximal topoisomerase II expression was tightly coupled to S phase and G2/M phase via both transcriptional and post-transcriptional regulation. Investigation in meiosis using pollen mother cells also revealed that it is not the major component of meiotic chromosomes, it seems to diffuse out once meiotic chromosomal condensation is completed. Synchronized tobacco BY-2 cell cultures were used to study the role of topoisomerase II in various stages of the cell cycle. Topoisomerase II transcript accumulation was observed during the S- and G2/M- phase of cell cycle. This biphasic expression pattern indicates the active requirement of topoisomerase II during these stages of the cell cycle. Through immuno-localization of topoisomerase II was observed diffusely throughout the nucleoplasm in interphase nuclei, whereas, the nucleolus region exhibited a more prominent immuno-positive staining that correlated with rRNA transcription, as shown by propidium iodide staining and BrUTP incorporation. The immuno-staining analysis also showed that topoisomerase II is the major component of mitotic chromosomes and remain attached to the chromosomes during cell division. The inhibition of topoisomerase II activity using specific inhibitors revealed quite dramatic effect on condensation of chromatin and chromosome individualization from prophase to metaphase transition. Partially condensed chromosomes were not arranged on metaphase plate and chromosomal perturbations were observed when advance to anaphase, suggesting the importance of topoisomerase II activity for proper chromosome condensation and segregation during mitosis. Contrary, topoisomerase II is not the major component of meiotic chromosomes, even though mitosis and meiosis share many processes, including the DNA replication, chromosome condensation and precisely regulated partitioning of chromosomes into daughter cells. Even if topoisomerase II is required for individualization and condensation of meiotic chromosomes, it seems to diffuse out once meiotic chromosomal condensation is completed.
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Affiliation(s)
- Badri Nath Singh
- Crop Improvement Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, Delhi, 110067, India
- Waksman Institute, Rutgers, The State University of New Jersey, Piscataway, NJ, USA
| | - V Mohan Murali Achary
- Crop Improvement Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, Delhi, 110067, India
| | - Varakumar Panditi
- Crop Improvement Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, Delhi, 110067, India
| | - Sudhir K Sopory
- Crop Improvement Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, Delhi, 110067, India
| | - Malireddy K Reddy
- Crop Improvement Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, Delhi, 110067, India.
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5
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Gambus A. Termination of Eukaryotic Replication Forks. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2017; 1042:163-187. [DOI: 10.1007/978-981-10-6955-0_8] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
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6
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Jacobsen RG, Mazloumi Gavgani F, Mellgren G, Lewis AE. DNA Topoisomerase IIα contributes to the early steps of adipogenesis in 3T3-L1 cells. Cell Signal 2016; 28:1593-603. [DOI: 10.1016/j.cellsig.2016.07.002] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/20/2016] [Accepted: 07/07/2016] [Indexed: 01/03/2023]
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Pedersen RT, Kruse T, Nilsson J, Oestergaard VH, Lisby M. TopBP1 is required at mitosis to reduce transmission of DNA damage to G1 daughter cells. J Cell Biol 2015; 210:565-82. [PMID: 26283799 PMCID: PMC4539992 DOI: 10.1083/jcb.201502107] [Citation(s) in RCA: 71] [Impact Index Per Article: 7.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
Genome integrity is critically dependent on timely DNA replication and accurate chromosome segregation. Replication stress delays replication into G2/M, which in turn impairs proper chromosome segregation and inflicts DNA damage on the daughter cells. Here we show that TopBP1 forms foci upon mitotic entry. In early mitosis, TopBP1 marks sites of and promotes unscheduled DNA synthesis. Moreover, TopBP1 is required for focus formation of the structure-selective nuclease and scaffold protein SLX4 in mitosis. Persistent TopBP1 foci transition into 53BP1 nuclear bodies (NBs) in G1 and precise temporal depletion of TopBP1 just before mitotic entry induced formation of 53BP1 NBs in the next cell cycle, showing that TopBP1 acts to reduce transmission of DNA damage to G1 daughter cells. Based on these results, we propose that TopBP1 maintains genome integrity in mitosis by controlling chromatin recruitment of SLX4 and by facilitating unscheduled DNA synthesis.
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Affiliation(s)
| | - Thomas Kruse
- The Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, DK-2200 Copenhagen N, Denmark
| | - Jakob Nilsson
- The Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, DK-2200 Copenhagen N, Denmark
| | - Vibe H Oestergaard
- Department of Biology, University of Copenhagen, DK-2200 Copenhagen N, Denmark
| | - Michael Lisby
- Department of Biology, University of Copenhagen, DK-2200 Copenhagen N, Denmark
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8
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Moreno SP, Bailey R, Campion N, Herron S, Gambus A. Polyubiquitylation drives replisome disassembly at the termination of DNA replication. Science 2014; 346:477-81. [PMID: 25342805 DOI: 10.1126/science.1253585] [Citation(s) in RCA: 154] [Impact Index Per Article: 14.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/17/2023]
Abstract
Resolution of replication forks during termination of DNA replication is essential for accurate duplication of eukaryotic genomes. Here we present evidence consistent with the idea that polyubiquitylation of a replisome component (Mcm7) leads to its disassembly at the converging terminating forks because of the action of the p97/VCP/Cdc48 protein remodeler. Using Xenopus laevis egg extract, we have shown that blocking polyubiquitylation results in the prolonged association of the active helicase with replicating chromatin. The Mcm7 subunit is the only component of the active helicase that we find polyubiquitylated during replication termination. The observed polyubiquitylation is followed by disassembly of the active helicase dependent on p97/VCP/Cdc48. Altogether, our data provide insight into the mechanism of replisome disassembly during eukaryotic DNA replication termination.
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Affiliation(s)
- Sara Priego Moreno
- School of Cancer Sciences, University of Birmingham, Vincent Drive, Birmingham B15 2TT, UK
| | - Rachael Bailey
- School of Cancer Sciences, University of Birmingham, Vincent Drive, Birmingham B15 2TT, UK
| | - Nicholas Campion
- School of Cancer Sciences, University of Birmingham, Vincent Drive, Birmingham B15 2TT, UK
| | - Suzanne Herron
- School of Cancer Sciences, University of Birmingham, Vincent Drive, Birmingham B15 2TT, UK
| | - Agnieszka Gambus
- School of Cancer Sciences, University of Birmingham, Vincent Drive, Birmingham B15 2TT, UK.
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9
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Murillo-Pineda M, Cabello-Lobato MJ, Clemente-Ruiz M, Monje-Casas F, Prado F. Defective histone supply causes condensin-dependent chromatin alterations, SAC activation and chromosome decatenation impairment. Nucleic Acids Res 2014; 42:12469-82. [PMID: 25300489 PMCID: PMC4227775 DOI: 10.1093/nar/gku927] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
The structural organization of chromosomes is essential for their correct function and dynamics during the cell cycle. The assembly of DNA into chromatin provides the substrate for topoisomerases and condensins, which introduce the different levels of superhelical torsion required for DNA metabolism. In particular, Top2 and condensin are directly involved in both the resolution of precatenanes that form during replication and the formation of the intramolecular loop that detects tension at the centromeric chromatin during chromosome biorientation. Here we show that histone depletion activates the spindle assembly checkpoint (SAC) and impairs sister chromatid decatenation, leading to chromosome mis-segregation and lethality in the absence of the SAC. We demonstrate that histone depletion impairs chromosome biorientation and activates the Aurora-dependent pathway, which detects tension problems at the kinetochore. Interestingly, SAC activation is suppressed by the absence of Top2 and Smc2, an essential component of condensin. Indeed, smc2-8 suppresses catenanes accumulation, mitotic arrest and growth defects induced by histone depletion at semi-permissive temperature. Remarkably, SAC activation by histone depletion is associated with condensin-mediated alterations of the centromeric chromatin. Therefore, our results reveal the importance of a precise interplay between histone supply and condensin/Top2 for pericentric chromatin structure, precatenanes resolution and centromere biorientation.
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Affiliation(s)
- Marina Murillo-Pineda
- Departamento de Biología Molecular, Centro Andaluz de Biología Molecular y Medicina Regenerativa (CABIMER), Consejo Superior de Investigaciones Científicas (CSIC), Seville, Spain
| | - María J Cabello-Lobato
- Departamento de Biología Molecular, Centro Andaluz de Biología Molecular y Medicina Regenerativa (CABIMER), Consejo Superior de Investigaciones Científicas (CSIC), Seville, Spain
| | - Marta Clemente-Ruiz
- Departamento de Biología Molecular, Centro Andaluz de Biología Molecular y Medicina Regenerativa (CABIMER), Consejo Superior de Investigaciones Científicas (CSIC), Seville, Spain
| | | | - Félix Prado
- Departamento de Biología Molecular, Centro Andaluz de Biología Molecular y Medicina Regenerativa (CABIMER), Consejo Superior de Investigaciones Científicas (CSIC), Seville, Spain
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10
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Bau JT, Kang Z, Austin CA, Kurz EU. Salicylate, a catalytic inhibitor of topoisomerase II, inhibits DNA cleavage and is selective for the α isoform. Mol Pharmacol 2014; 85:198-207. [PMID: 24220011 DOI: 10.1124/mol.113.088963] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/14/2025] Open
Abstract
Topoisomerase II (topo II) is a ubiquitous enzyme that is essential for cell survival through its role in regulating DNA topology and chromatid separation. Topo II can be poisoned by common chemotherapeutics (such as doxorubicin and etoposide), leading to the accumulation of cytotoxic enzyme-linked DNA double-stranded breaks. In contrast, nonbreak-inducing topo II catalytic inhibitors have also been described and have more limited use in clinical chemotherapy. These agents, however, may alter the efficacy of regimens incorporating topo II poisons. We previously identified salicylate, the primary metabolite of aspirin, as a novel catalytic inhibitor of topo II. We have now determined the mechanism by which salicylate inhibits topo II. As catalytic inhibitors can act at a number of steps in the topo II catalytic cycle, we used multiple independent, biochemical approaches to interrogate the catalytic cycle. Furthermore, as mammalian cells express two isoforms of topo II (α and β), we examined whether salicylate was isoform selective. Our results demonstrate that salicylate is unable to intercalate DNA, and does not prevent enzyme-DNA interaction, nor does it promote stabilization of topo IIα in closed clamps on DNA. Although salicylate decreased topo IIα ATPase activity in a dose-dependent noncompetitive manner, this was secondary to salicylate-mediated inhibition of DNA cleavage. Surprisingly, comparison of salicylate's effects using purified human topo IIα and topo IIβ revealed that salicylate selectively inhibits the α isoform. These findings provide a definitive mechanism for salicylate-mediated inhibition of topo IIα and provide support for further studies determining the basis for its isoform selectivity.
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Affiliation(s)
- Jason T Bau
- Southern Alberta Cancer Research Institute and Department of Physiology and Pharmacology, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada (J.T.B., Z.K., E.U.K.); and Institute for Cellular and Molecular Biosciences, Newcastle University, Newcastle upon Tyne, Tyne and Wear, United Kingdom (C.A.A.)
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11
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Farr CJ, Antoniou-Kourounioti M, Mimmack ML, Volkov A, Porter ACG. The α isoform of topoisomerase II is required for hypercompaction of mitotic chromosomes in human cells. Nucleic Acids Res 2014; 42:4414-26. [PMID: 24476913 PMCID: PMC3985649 DOI: 10.1093/nar/gku076] [Citation(s) in RCA: 36] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/17/2023] Open
Abstract
As proliferating cells transit from interphase into M-phase, chromatin undergoes extensive reorganization, and topoisomerase (topo) IIα, the major isoform of this enzyme present in cycling vertebrate cells, plays a key role in this process. In this study, a human cell line conditional null mutant for topo IIα and a derivative expressing an auxin-inducible degron (AID)-tagged version of the protein have been used to distinguish real mitotic chromosome functions of topo IIα from its more general role in DNA metabolism and to investigate whether topo IIβ makes any contribution to mitotic chromosome formation. We show that topo IIβ does contribute, with endogenous levels being sufficient for the initial stages of axial shortening. However, a significant effect of topo IIα depletion, seen with or without the co-depletion of topo IIβ, is the failure of chromosomes to hypercompact when delayed in M-phase. This requires much higher levels of topo II protein and is impaired by drugs or mutations that affect enzyme activity. A prolonged delay at the G2/M border results in hyperefficient axial shortening, a process that is topo IIα-dependent. Rapid depletion of topo IIα has allowed us to show that its function during late G2 and M-phase is truly required for shaping mitotic chromosomes.
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Affiliation(s)
- Christine J Farr
- Department of Genetics, University of Cambridge, Downing St, Cambridge CB2 3EH, UK and Centre for Haematology, Faculty of Medicine, Imperial College London, Hammersmith Hospital Campus, Du Cane Rd, London W12 0NN, UK
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12
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Kurosawa A, Saito S, So S, Hashimoto M, Iwabuchi K, Watabe H, Adachi N. DNA ligase IV and artemis act cooperatively to suppress homologous recombination in human cells: implications for DNA double-strand break repair. PLoS One 2013; 8:e72253. [PMID: 23967291 PMCID: PMC3743779 DOI: 10.1371/journal.pone.0072253] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/18/2013] [Accepted: 07/08/2013] [Indexed: 11/24/2022] Open
Abstract
Nonhomologous end-joining (NHEJ) and homologous recombination (HR) are two major pathways for repairing DNA double-strand breaks (DSBs); however, their respective roles in human somatic cells remain to be elucidated. Here we show using a series of human gene-knockout cell lines that NHEJ repairs nearly all of the topoisomerase II- and low-dose radiation-induced DNA damage, while it negatively affects survival of cells harbouring replication-associated DSBs. Intriguingly, we find that loss of DNA ligase IV, a critical NHEJ ligase, and Artemis, an NHEJ factor with endonuclease activity, independently contribute to increased resistance to replication-associated DSBs. We also show that loss of Artemis alleviates hypersensitivity of DNA ligase IV-null cells to low-dose radiation- and topoisomerase II-induced DSBs. Finally, we demonstrate that Artemis-null human cells display increased gene-targeting efficiencies, particularly in the absence of DNA ligase IV. Collectively, these data suggest that DNA ligase IV and Artemis act cooperatively to promote NHEJ, thereby suppressing HR. Our results point to the possibility that HR can only operate on accidental DSBs when NHEJ is missing or abortive, and Artemis may be involved in pathway switching from incomplete NHEJ to HR.
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Affiliation(s)
- Aya Kurosawa
- Graduate School of Nanobioscience, Yokohama City University, Yokohama, Japan
| | - Shinta Saito
- Graduate School of Nanobioscience, Yokohama City University, Yokohama, Japan
| | - Sairei So
- Graduate School of Nanobioscience, Yokohama City University, Yokohama, Japan
| | | | - Kuniyoshi Iwabuchi
- Department of Biochemistry, Kanazawa Medical University, Ishikawa, Japan
| | - Haruka Watabe
- Graduate School of Nanobioscience, Yokohama City University, Yokohama, Japan
| | - Noritaka Adachi
- Graduate School of Nanobioscience, Yokohama City University, Yokohama, Japan
- Advanced Medical Research Center, Yokohama City University, Yokohama, Japan
- * E-mail:
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13
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Gaggioli V, Le Viet B, Germe T, Hyrien O. DNA topoisomerase IIα controls replication origin cluster licensing and firing time in Xenopus egg extracts. Nucleic Acids Res 2013; 41:7313-31. [PMID: 23757188 PMCID: PMC3753627 DOI: 10.1093/nar/gkt494] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/19/2022] Open
Abstract
Sperm chromatin incubated in Xenopus egg extracts undergoes origin licensing and nuclear assembly before DNA replication. We found that depletion of DNA topoisomerase IIα (topo IIα), the sole topo II isozyme of eggs and its inhibition by ICRF-193, which clamps topo IIα around DNA have opposite effects on these processes. ICRF-193 slowed down replication origin cluster activation and fork progression in a checkpoint-independent manner, without altering replicon size. In contrast, topo IIα depletion accelerated origin cluster activation, and topo IIα add-back negated overinitiation. Therefore, topo IIα is not required for DNA replication, but topo IIα clamps slow replication, probably by forming roadblocks. ICRF-193 had no effect on DNA synthesis when added after nuclear assembly, confirming that topo IIα activity is dispensable for replication and revealing that topo IIα clamps formed on replicating DNA do not block replication, presumably because topo IIα acts behind and not in front of forks. Topo IIα depletion increased, and topo IIα addition reduced, chromatin loading of MCM2-7 replicative helicase, whereas ICRF-193 did not affect MCM2-7 loading. Therefore, topo IIα restrains MCM2-7 loading in an ICRF-193-resistant manner during origin licensing, suggesting a model for establishing the sequential firing of origin clusters.
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Affiliation(s)
- Vincent Gaggioli
- Institut de Biologie de l'Ecole Normale Supérieure (IBENS), S2-Génomique Fonctionnelle, CNRS UMR8197, Inserm U1024, 46 rue d'Ulm, 75005 Paris, France
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14
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Rao VA. Iron chelators with topoisomerase-inhibitory activity and their anticancer applications. Antioxid Redox Signal 2013; 18:930-55. [PMID: 22900902 PMCID: PMC3557438 DOI: 10.1089/ars.2012.4877] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Abstract
SIGNIFICANCE Iron and topoisomerases are abundant and essential cellular components. Iron is required for several key processes such as DNA synthesis, mitochondrial electron transport, synthesis of heme, and as a co-factor for many redox enzymes. Topoisomerases serve as critical enzymes that resolve topological problems during DNA synthesis, transcription, and repair. Neoplastic cells have higher uptake and utilization of iron, as well as elevated levels of topoisomerase family members. Separately, the chelation of iron and the cytotoxic inhibition of topoisomerase have yielded potent anticancer agents. RECENT ADVANCES The chemotherapeutic drugs doxorubicin and dexrazoxane both chelate iron and target topoisomerase 2 alpha (top2α). Newer chelators such as di-2-pyridylketone-4,4,-dimethyl-3-thiosemicarbazone and thiosemicarbazone -24 have recently been identified as top2α inhibitors. The growing list of agents that appear to chelate iron and inhibit topoisomerases prompts the question of whether and how these two distinct mechanisms might interplay for a cytotoxic chemotherapeutic outcome. CRITICAL ISSUES While iron chelation and topoisomerase inhibition each represent mechanistically advantageous anticancer therapeutic strategies, dual targeting agents present an attractive multi-modal opportunity for enhanced anticancer tumor killing and overcoming drug resistance. The commonalities and caveats of dual inhibition are presented in this review. FUTURE DIRECTIONS Gaps in knowledge, relevant biomarkers, and strategies for future in vivo studies with dual inhibitors are discussed.
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Affiliation(s)
- V Ashutosh Rao
- Laboratory of Biochemistry, Division of Therapeutic Proteins, Office of Biotechnology Products, Office of Pharmaceutical Science, Center for Drug Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892, USA.
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16
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Topoisomerase II binds nucleosome-free DNA and acts redundantly with topoisomerase I to enhance recruitment of RNA Pol II in budding yeast. Proc Natl Acad Sci U S A 2011; 108:12693-8. [PMID: 21771901 DOI: 10.1073/pnas.1106834108] [Citation(s) in RCA: 64] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
DNA topoisomerases are believed to promote transcription by removing excessive DNA supercoils produced during elongation. However, it is unclear how topoisomerases in eukaryotes are recruited and function in the transcription pathway in the context of nucleosomes. To address this problem we present high-resolution genome-wide maps of one of the major eukaryotic topoisomerases, Topoisomerase II (Top2) and nucleosomes in the budding yeast, Saccharomyces cerevisiae. Our data indicate that at promoters Top2 binds primarily to DNA that is nucleosome-free. However, although nucleosome loss enables Top2 occupancy, the opposite is not the case and the loss of Top2 has little effect on nucleosome density. We also find that Top2 is involved in transcription. Not only is Top2 enriched at highly transcribed genes, but Top2 is required redundantly with Top1 for optimal recruitment of RNA polymerase II at their promoters. These findings and the examination of candidate-activated genes suggest that nucleosome loss induced by nucleosome remodeling factors during gene activation enables Top2 binding, which in turn acts redundantly with Top1 to enhance recruitment of RNA polymerase II.
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17
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Le H, Singh S, Shih SJ, Du N, Schnyder S, Loredo GA, Bien C, Michaelis L, Toor A, Diaz MO, Vaughan AT. Rearrangements of the MLL gene are influenced by DNA secondary structure, potentially mediated by topoisomerase II binding. Genes Chromosomes Cancer 2009; 48:806-15. [PMID: 19530238 DOI: 10.1002/gcc.20685] [Citation(s) in RCA: 32] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
The location of MLL translocation breakpoints within therapy-related acute myeloid leukemia linked to drugs targeting Topoisomerase II and infant acute leukemia (IAL) are biased toward the intron 11-exon 12 region of MLL, although lacking a comprehensive explanation. To address this, blood samples were taken from breast cancer and lymphoma patients receiving Topoisomerase II inhibitor therapy. Inverse PCR analysis was used to interrogate the exon 12 region of MLL for rearrangements. Eleven of 19 observed translocations showed breakpoint junctions restricted to a single 5 bp location within exon 12. A similarly restricted distribution (11/20 breakpoint junctions) was observed in TK6 cells exposed to either estrogen (linked to IAL) or anti-CD95 antibody. The translocation hotspot was at the 5' edge of a 10-bp tract matched with a perfect palindrome, 101 bp distant. A high stringency Topoisomerase II consensus sequence binding site was noted at the geometric midpoint of the palindromes. Ligation-mediated PCR to screen TK6 cells exposed to anti-CD95 antibody showed 14/37 (38%) of DNA breaks adjacent to the 5' palindrome and 10/37 (27%) at the 3' partner. We propose a model whereby Topoisomerase II facilitates the organization of nuclease-sensitive secondary structures, stabilized by palindrome association, which are prone to rearrangement.
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Affiliation(s)
- Hongan Le
- Department of Radiation Oncology, UC Davis Cancer Center, Sacramento, CA 95817, USA
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18
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Martínez-Robles ML, Witz G, Hernández P, Schvartzman JB, Stasiak A, Krimer DB. Interplay of DNA supercoiling and catenation during the segregation of sister duplexes. Nucleic Acids Res 2009; 37:5126-37. [PMID: 19553196 PMCID: PMC2731910 DOI: 10.1093/nar/gkp530] [Citation(s) in RCA: 35] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
Abstract
The discrete regulation of supercoiling, catenation and knotting by DNA topoisomerases is well documented both in vivo and in vitro, but the interplay between them is still poorly understood. Here we studied DNA catenanes of bacterial plasmids arising as a result of DNA replication in Escherichia coli cells whose topoisomerase IV activity was inhibited. We combined high-resolution two-dimensional agarose gel electrophoresis with numerical simulations in order to better understand the relationship between the negative supercoiling of DNA generated by DNA gyrase and the DNA interlinking resulting from replication of circular DNA molecules. We showed that in those replication intermediates formed in vivo, catenation and negative supercoiling compete with each other. In interlinked molecules with high catenation numbers negative supercoiling is greatly limited. However, when interlinking decreases, as required for the segregation of newly replicated sister duplexes, their negative supercoiling increases. This observation indicates that negative supercoiling plays an active role during progressive decatenation of newly replicated DNA molecules in vivo.
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Affiliation(s)
- María Luisa Martínez-Robles
- Departamento de Biología Celular y del Desarrollo, Centro de Investigaciones Biológicas (CSIC), Ramiro de Maeztu 9, 28040 Madrid, Spain
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19
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Johnson M, Phua HH, Bennett SC, Spence JM, Farr CJ. Studying vertebrate topoisomerase 2 function using a conditional knockdown system in DT40 cells. Nucleic Acids Res 2009; 37:e98. [PMID: 19494182 PMCID: PMC2724289 DOI: 10.1093/nar/gkp480] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022] Open
Abstract
DT40 is a B-cell lymphoma-derived avian cell line widely used to study cell autonomous gene function because of the high rates with which DNA constructs are homologously recombined into its genome. Here, we demonstrate that the power of the DT40 system can be extended yet further through the use of RNA interference as an alternative to gene targeting. We have generated and characterized stable DT40 transfectants in which both topo 2 genes have been in situ tagged using gene targeting, and from which the mRNA of both topoisomerase 2 isoforms can be conditionally depleted through the tetracycline-induced expression of short hairpin RNAs. The cell cycle phenotype of topo 2-depleted DT40 cells has been compared with that previously reported for other vertebrate cells depleted either of topo 2α through gene targeting, or depleted of both isoforms simultaneously by transient RNAi. In addition, the DT40 knockdown system has been used to explore whether excess catenation arising through topo 2 depletion is sufficient to trigger the G2 catenation (or decatenation) checkpoint, proposed to exist in differentiated vertebrate cells.
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Affiliation(s)
- Mark Johnson
- Department of Genetics, University of Cambridge, Downing St, Cambridge CB2 3EH, UK
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Abstract
Recent molecular studies have expanded the biological contexts in which topoisomerase II (TOP2) has crucial functions, including DNA replication, transcription and chromosome segregation. Although the biological functions of TOP2 are important for ensuring genomic integrity, the ability to interfere with TOP2 and generate enzyme-mediated DNA damage is an effective strategy for cancer chemotherapy. The molecular tools that have allowed an understanding of the biological functions of TOP2 are also being applied to understanding the details of drug action. These studies promise refined targeting of TOP2 as an effective anticancer strategy.
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Affiliation(s)
- John L Nitiss
- Molecular Pharmacology Department, St Jude Children's Research Hospital, Memphis, TN 38105, USA.
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21
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Abstract
The nucleotide sequence of DNA is the repository of hereditary information. Yet, it is now clear that the DNA itself plays an active role in regulating the ability of the cell to extract its information. Basic biological processes, including control of gene transcription, faithful DNA replication and segregation, maintenance of the genome and cellular differentiation are subject to the conformational and topological properties of DNA in addition to the regulation imparted by the sequence itself. How do these DNA features manifest such striking effects and how does the cell regulate them? In this review, we describe how misregulation of DNA topology can lead to cellular dysfunction. We then address how cells prevent these topological problems. We close with a discussion on recent theoretical advances indicating that the topological problems, themselves, can provide the cues necessary for their resolution by type-2 topoisomerases.
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Affiliation(s)
- Zhirong Liu
- College of Chemistry and Molecular Engineering, and Center for Theoretical Biology, Peking University, Beijing 100871, China
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22
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Lee MT, Bachant J. SUMO modification of DNA topoisomerase II: trying to get a CENse of it all. DNA Repair (Amst) 2009; 8:557-68. [PMID: 19230795 DOI: 10.1016/j.dnarep.2009.01.004] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/29/2022]
Abstract
DNA topoisomerase II (topo II) is an essential determinant of chromosome structure and function, acting to resolve topological problems inherent in recombining, transcribing, replicating and segregating DNA. In particular, the unique decatenating activity of topo II is required for sister chromatids to disjoin and separate in mitosis. Topo II exhibits a dynamic localization pattern on mitotic chromosomes, accumulating at centromeres and axial chromosome cores prior to anaphase. In organisms ranging from yeast to humans, a fraction of topo II is targeted for SUMO conjugation in mitotic cells, and here we review our current understanding of the significance of this modification. As we shall see, an emerging consensus is that in metazoans SUMO modification is required for topo II to accumulate at centromeres, and that in the absence of this regulation there is an elevated frequency of chromosome non-disjunction, segregation errors, and aneuploidy. The underlying molecular mechanisms for how SUMO controls topo II are as yet unclear. In closing, however, we will evaluate two possible interpretations: one in which SUMO promotes enzyme turnover, and a second in which SUMO acts as a localization tag for topo II chromosome trafficking.
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Affiliation(s)
- Ming-Ta Lee
- Department of Cell Biology and Neuroscience, University of California, Riverside, Riverside, CA 92521, USA
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23
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Labit H, Perewoska I, Germe T, Hyrien O, Marheineke K. DNA replication timing is deterministic at the level of chromosomal domains but stochastic at the level of replicons in Xenopus egg extracts. Nucleic Acids Res 2008; 36:5623-34. [PMID: 18765475 PMCID: PMC2553594 DOI: 10.1093/nar/gkn533] [Citation(s) in RCA: 37] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023] Open
Abstract
Replication origins in Xenopus egg extracts are located at apparently random sequences but are activated in clusters that fire at different times during S phase under the control of ATR/ATM kinases. We investigated whether chromosomal domains and single sequences replicate at distinct times during S phase in egg extracts. Replication foci were found to progressively appear during early S phase and foci labelled early in one S phase colocalized with those labelled early in the next S phase. However, the distribution of these two early labels did not coincide between single origins or origin clusters on single DNA fibres. The 4 Mb Xenopus rDNA repeat domain was found to replicate later than the rest of the genome and to have a more nuclease-resistant chromatin structure. Replication initiated more frequently in the transcription unit than in the intergenic spacer. These results suggest for the first time that in this embryonic system, where transcription does not occur, replication timing is deterministic at the scale of large chromatin domains (1–5 Mb) but stochastic at the scale of replicons (10 kb) and replicon clusters (50–100 kb).
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Affiliation(s)
- Hélène Labit
- Ecole Normale Supérieure, Biology Department, Laboratory of Molecular Genetics, CNRS UMR 8541, 46, rue d'Ulm, 75005 Paris, France
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24
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A novel peroxisome proliferator-activated receptor alpha/gamma agonist, BPR1H0101, inhibits topoisomerase II catalytic activity in human cancer cells. Anticancer Drugs 2008; 19:151-8. [PMID: 18176111 DOI: 10.1097/cad.0b013e3282f28fe] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
Abstract
Peroxisome proliferator-activated receptor (PPAR) gamma agonists are used clinically for treating diabetes mellitus and cancer. 2-Methyl-2[(1-{3-phenyl-7-propylbenzol[d]isoxazol-6-yl}oxy)propyl]-1H-4-indolyl) oxy]propanoic acid (BPR1H0101) is a novel synthetic indole-based compound, discovered through research to identify new PPARgamma agonists, and it acts as a dual agonist for PPARgamma and PPARalpha. Isobologram analysis demonstrated that BPR1H0101 is capable of antagonistic interaction with the topoisomerase (topo) II poison, VP16. A study of its mechanism showed that BPR1H0101 could inhibit the catalytic activity of topo II in vitro, but did not produce detectable topo II-mediated DNA strand breaks in human oral cancer KB cells. Furthermore, BPR1H0101 could inhibit VP16-induced topo II-mediated DNA cleavage and ataxia-telangiectasia-mutated phosphorylation in KB cells. The results suggest that BPR1H0101 can interfere with the topo II reaction by inhibiting catalytic activity before the formation of the intermediate cleavable complex; consequently, it can impede VP16-induced topo II-mediated DNA cleavage and cell death. This is the first identified PPARalpha/gamma agonist that can serve as a topo II catalytic inhibitor, to interfere with VP16-induced cell death. The result might have relevance to the clinical use of the PPARalpha/gamma agonist in combination chemotherapy.
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25
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Wang L, Roy SK, Eastmond DA. Differential cell cycle-specificity for chromosomal damage induced by merbarone and etoposide in V79 cells. Mutat Res 2006; 616:70-82. [PMID: 17174356 DOI: 10.1016/j.mrfmmm.2006.11.023] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022]
Abstract
Merbarone, a topoisomerase II (topo II) inhibitor which, in contrast to etoposide, does not stabilize topo II-DNA cleavable complexes, was previously shown to be a potent clastogen in vitro and in vivo. To investigate the possible mechanisms, we compared the cell cycle-specificity of the clastogenic effects of merbarone and etoposide in V79 cells. Using flow cytometry and BrdU labeling techniques, etoposide was shown to cause a rapid and persistent G2 delay while merbarone was shown to cause a prolonged S-phase followed by a G2 delay. To identify the stages which are susceptible to DNA damage, we performed the micronucleus (MN) assay with synchronized cells or utilized a combination of BrdU pulse labeling and the cytokinesis-blocked MN assay with non-synchronized cells. Treatment of M phase cells with either agent did not result in increased MN formation. Etoposide but not merbarone caused a significant increase in MN when cells were treated during G2 phase. When treated during S-phase, both chemicals induced highly significant increases in MN. However, the relative proportion of MN induced by merbarone was substantially higher than that induced by etoposide. Both chemicals also caused significant increases in MN in cells that were treated during G1 phase. To confirm the observations in the MN assay, first division metaphases were evaluated in the chromosome aberration assay. The chromosomes of cells treated with merbarone and etoposide showed increased frequencies of both chromatid- and chromosome-type of aberrations. Our findings indicate that while etoposide causes DNA damage more evenly throughout the G1, S and G2 phases of the cell cycle, an outcome which may be closely associated with topo II-mediated DNA strand cleavage, merbarone induces DNA breakage primarily during S-phase, an effect which is likely due to the stalling of replication forks by inhibition of topo II activity.
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Affiliation(s)
- Ling Wang
- Environmental Toxicology Graduate Program, 2109 Biological Sciences Building, University of California, Riverside, CA 92521, USA
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26
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Liu Z, Zechiedrich EL, Chan HS. Inferring global topology from local juxtaposition geometry: interlinking polymer rings and ramifications for topoisomerase action. Biophys J 2006; 90:2344-55. [PMID: 16537549 PMCID: PMC1403174 DOI: 10.1529/biophysj.105.076778] [Citation(s) in RCA: 32] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
Lattice modeling is applied to investigate how the configurations of local chain juxtapositions may provide information about whether two ring polymers (loops) are topologically linked globally. Given a particular juxtaposition, the conditional probability that the loops are linked is determined by exact enumeration and extensive Monte Carlo sampling of conformations satisfying excluded volume constraints. A discrimination factor fL, defined as the ratio of linked to unlinked probabilities, varies widely depending on which juxtaposition is presumed. /log fL/s that are large for small loop size n tend to decrease, signaling diminishing topological information content of the juxtapositions, with increasing n. However, some juxtaposition geometries can impose sufficient overall conformational biases such that /log fL/ remains significant for large n. Notably, for two loops as large as n=200 in the model, the probability that passing the segments of a hooked juxtaposition would unlink an originally linked configuration is remarkably high, approximately 85%. In contrast, segment-passage of a free juxtaposition would link the loops from an originally unlinked configuration more than 90% of the time. The statistical mechanical principles emerging from these findings suggest that it is physically possible for DNA topoisomerases to decatenate effectively by acting selectively on juxtapositions with specific "hooked" geometries.
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Affiliation(s)
- Zhirong Liu
- Department of Biochemistry, Faculty of Medicine, University of Toronto, Toronto, Ontario M5S 1A8, Canada
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27
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Liu Z, Mann JK, Zechiedrich EL, Chan HS. Topological information embodied in local juxtaposition geometry provides a statistical mechanical basis for unknotting by type-2 DNA topoisomerases. J Mol Biol 2006; 361:268-85. [PMID: 16842819 DOI: 10.1016/j.jmb.2006.06.005] [Citation(s) in RCA: 46] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/13/2006] [Revised: 06/01/2006] [Accepted: 06/03/2006] [Indexed: 10/24/2022]
Abstract
Topoisomerases may unknot by recognizing specific DNA juxtapositions. The physical basis of this hypothesis is investigated by considering single-loop conformations in a coarse-grained polymer model. We determine the statistical relationship between the local geometry of a juxtaposition of two chain segments and whether the loop is knotted globally, and ascertain how the knot/unknot topology is altered by a topoisomerase-like segment passage at the juxtaposition. Segment passages at a "free" juxtaposition tend to increase knot probability. In contrast, segment passages at a "hooked" juxtaposition cause more transitions from knot to unknot than vice versa, resulting in a steady-state knot probability far lower than that at topological equilibrium. The reduction in knot population by passing chain segments through a hooked juxtaposition is more prominent for loops of smaller sizes, n, but remains significant even for larger loops: steady-state knot probability is only approximately 2%, and approximately 5% of equilibrium, respectively, for n=100 and 500 in the model. An exhaustive analysis of approximately 6000 different juxtaposition geometries indicates that the ability of a segment passage to unknot correlates strongly with the juxtaposition's "hookedness". Remarkably, and consistent with experiments on type-2 topoisomerases from different organisms, the unknotting potential of a juxtaposition geometry in our polymer model correlates almost perfectly with its corresponding decatenation potential. These quantitative findings suggest that it is possible for topoisomerases to disentangle by acting selectively on juxtapositions with "hooked" geometries.
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Affiliation(s)
- Zhirong Liu
- Department of Biochemistry, and Department of Medical Genetics and Microbiology, Faculty of Medicine, University of Toronto, Toronto, Ontario M5S 1A8, Canada
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Salceda J, Fernández X, Roca J. Topoisomerase II, not topoisomerase I, is the proficient relaxase of nucleosomal DNA. EMBO J 2006; 25:2575-83. [PMID: 16710299 PMCID: PMC1478187 DOI: 10.1038/sj.emboj.7601142] [Citation(s) in RCA: 83] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/21/2006] [Accepted: 04/18/2006] [Indexed: 11/09/2022] Open
Abstract
Eukaryotic topoisomerases I and II efficiently remove helical tension in naked DNA molecules. However, this activity has not been examined in nucleosomal DNA, their natural substrate. Here, we obtained yeast minichromosomes holding DNA under (+) helical tension, and incubated them with topoisomerases. We show that DNA supercoiling density can rise above +0.04 without displacement of the histones and that the typical nucleosome topology is restored upon DNA relaxation. However, in contrast to what is observed in naked DNA, topoisomerase II relaxes nucleosomal DNA much faster than topoisomerase I. The same effect occurs in cell extracts containing physiological dosages of topoisomeraseI and II. Apparently, the DNA strand-rotation mechanism of topoisomerase I does not efficiently relax chromatin, which imposes barriers for DNA twist diffusion. Conversely, the DNA cross-inversion mechanism of topoisomerase II is facilitated in chromatin, which favor the juxtaposition of DNA segments. We conclude that topoisomerase II is the main modulator of DNA topology in chromatin fibers. The nonessential topoisomerase I then assists DNA relaxation where chromatin structure impairs DNA juxtaposition but allows twist diffusion.
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Affiliation(s)
- Javier Salceda
- Institut de Biología Molecular de Barcelona, CSIC, Barcelona, Spain
| | - Xavier Fernández
- Institut de Biología Molecular de Barcelona, CSIC, Barcelona, Spain
| | - Joaquim Roca
- Institut de Biología Molecular de Barcelona, CSIC, Barcelona, Spain
- Institut de Biología Molecular de Barcelona, CSIC, Jordi Girona 18-26, 08034 Barcelona, Spain. Tel.: 34 934 006 178; Fax: 34 932 045 904; E-mail:
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Randall GL, Pettitt BM, Buck GR, Zechiedrich EL. Electrostatics of DNA-DNA juxtapositions: consequences for type II topoisomerase function. JOURNAL OF PHYSICS. CONDENSED MATTER : AN INSTITUTE OF PHYSICS JOURNAL 2006; 18:S173-S185. [PMID: 19088861 PMCID: PMC2603137 DOI: 10.1088/0953-8984/18/14/s03] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/22/2023]
Abstract
Type II topoisomerases resolve problematic DNA topologies such as knots, catenanes, and supercoils that arise as a consequence of DNA replication and recombination. Failure to remove problematic DNA topologies prohibits cell division and can result in cell death or genetic mutation. Such catastrophic consequences make topoisomerases an effective target for antibiotics and anticancer agents. Despite their biological and clinical importance, little is understood about how a topoisomerase differentiates DNA topologies in a molecule that is significantly larger than the topoisomerase itself. It has been proposed that type II topoisomerases recognize angle and curvature between two DNA helices characteristic of knotted and catenated DNA to account for the enzyme's preference to unlink instead of link DNA. Here we consider the electrostatic potential of DNA juxtapositions to determine the possibility of juxtapositions occurring through Brownian diffusion. We found that despite the large negative electrostatic potential formed between two juxtaposed DNA helices, a bulk counterion concentration as small as 50 mM provides sufficient electrostatic screening to prohibit significant interaction beyond an interhelical separation of 3 nm in both hooked and free juxtapositions. This suggests that instead of electrostatics, mechanical forces such as those occurring in anaphase, knots, catenanes, or the writhe of supercoiled DNA may be responsible for the formation of DNA juxtapositions.
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Affiliation(s)
- Graham L Randall
- Program in Structural and Computational Biology and Molecular Biophysics, Baylor College of Medicine, Houston, TX 77030, USA
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