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Andrzej Skladanowski - One of the best experts on this subject based on the ideXlab platform.
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Novel anticancer strategy aimed at targeting Shelterin complexes by the induction of structural changes in telomeric DNA: hitting two birds with one stone.
Current cancer drug targets, 2014Co-Authors: Joanna Bidzinska, Maciej Baginski, Andrzej SkladanowskiAbstract:The ends of chromosomes in mammals are composed of telomeric DNA containing TTAGGG repeats, which bind specific proteins called Shelterins. This telomeric DNA together with Shelterins form a cap that protects the ends of chromosomes from being recognized as sites of DNA damage and from chromosomal fusions. Many very successful antitumor drugs used in the treatment of cancer patients bind to DNA, some of them with a prominent sequence specificity leads to changes in DNA structure and integrity. We propose a new target for antitumor drugs where small molecule ligands can bind to telomeric DNA and induce specific structural changes. These changes would lead to a selective interference with the formation of telomeric DNA-Shelterin complexes, especially involving TRF1 and TRF2 proteins, as these proteins bind double-stranded telomeric DNA in a sequence- and structure-dependent manner. The rationale of the proposed therapeutic strategy is further justified by the fact that tumor cells have relatively short telomeres and frequently de-regulated Shelterin expression and/or functionality. Thus uncapping of chromosome ends by DNA binding compounds which disrupt DNA-Shelterin complexes can ultimately induce selective cytotoxic effect in tumor cells. Possible implications for rational design of new antitumor drugs which interfere with telomeric DNA structure and formation of DNA-Shelterin complexes are discussed.
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Abstract C159: Specific structural changes in telomeric DNA induced by triazoloacridone compound C‐1305 prevent the formation of TRF1/2 Shelterin complexes
Molecular Cancer Therapeutics, 2009Co-Authors: Joanna Bidzinska, Maciej Baginski, Marcin Wojciechowski, Andrzej SkladanowskiAbstract:C‐1305 is a triazoloacridone derivative with potent activity in lung and colon cancer models. We reported previously that compound C‐1305 binds to DNA by intercalation and induces structural distortions in DNA regions containing guanine triplets GGG, at concentrations as low as 0.1 µM. This is a unique property of C‐1305 since similar results were not observed for any of the structurally related triazoloacridone derivatives tested or other DNA binding compounds. Human telomeres consist of tandem repeats of the TTAGGG fragments and form specialized DNA‐protein complexes consisting of telomere binding proteins (Shelterins), localized at the ends of the linear chromosomes. Most of telomeric DNA is double‐stranded except the most extreme part where 3′ region of the G strand is single‐stranded and may form the so‐called G‐quadruplex structures. Two telomere‐binding proteins, TRF1 and TRF2, are the key components of the Shelterin complex that bind double‐stranded telomeric DNA. Both TRF1 and TRF2 contain the so‐called Myb‐like domain, which is responsible for sequence‐specific binding of these proteins to DNA. Previous studies have shown that the absence or non‐functionality of either of TRF proteins results in increased telomere length and chromosomal fusions that ultimately leads to cell death. In this study, we wanted to clarify whether compound C‐1305 and its structural analogs bind telomeric DNA and stabilize G‐quadruplexes. The binding affinities of studied compounds to telomeric DNA were tested against different DNA substrates using microdialysis and DNA melting temperature assays and stabilization of G‐quadruplexes by FRET assay. Given that telomeric repeats contain GGG triplets, we also wanted to assess whether C‐1305 induces structural distortions within telomeric sequences and whether drug‐DNA binding changes the affinity of TRF1 and TRF2 to telomeric DNA. We show here that from all studied compounds C‐1305 showed the highest affinity and specificity to double‐stranded oligonucleotides containing telomeric TTAGGG repeats and to a oligonucleotide folded into intramolecular G‐quadruplex structure. Based on the molecular modeling studies, we also propose a model of C‐1305/G‐quadruplex complex. We recently postulated, that TRF1/2‐telomeric DNA complexes may be excellent targets for anticancer drugs, which by specific binding to telomeric DNA disturb the formation of Shelterin‐DNA complexes. In the second part of this work, we characterized interactions of recombinant TRF1 and TRF2 proteins and Myb‐like domain fragments, with telomeric DNA, in the absence or presence of compound C‐1305 and its analogs. Using chemical probing, we found that C‐1305 induces structural changes within GGG triplets present in the telomeric sequences in double‐stranded DNA substrate. We also showed by the EMSA assay that compound C‐1305 is disturbs the binding of TRF1/2 proteins and Myb‐domain peptide with telomeric DNA probes, containing consensus binding sequences for both studied Shelterins. Together, our results show that compound C‐1305 specifically binds both G‐quadruplex and double‐stranded telomeric DNA. Moreover, this compound induces structural changes in double stranded telomeric DNA, specifically within GGG triplets, that leads to greatly decreased affinity of both TRF1 and TRF2 to telomeric DNA substrates. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C159.
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abstract c159 specific structural changes in telomeric dna induced by triazoloacridone compound c 1305 prevent the formation of trf1 2 Shelterin complexes
Molecular Cancer Therapeutics, 2009Co-Authors: Joanna Bidzinska, Maciej Baginski, Marcin Wojciechowski, Andrzej SkladanowskiAbstract:C‐1305 is a triazoloacridone derivative with potent activity in lung and colon cancer models. We reported previously that compound C‐1305 binds to DNA by intercalation and induces structural distortions in DNA regions containing guanine triplets GGG, at concentrations as low as 0.1 µM. This is a unique property of C‐1305 since similar results were not observed for any of the structurally related triazoloacridone derivatives tested or other DNA binding compounds. Human telomeres consist of tandem repeats of the TTAGGG fragments and form specialized DNA‐protein complexes consisting of telomere binding proteins (Shelterins), localized at the ends of the linear chromosomes. Most of telomeric DNA is double‐stranded except the most extreme part where 3′ region of the G strand is single‐stranded and may form the so‐called G‐quadruplex structures. Two telomere‐binding proteins, TRF1 and TRF2, are the key components of the Shelterin complex that bind double‐stranded telomeric DNA. Both TRF1 and TRF2 contain the so‐called Myb‐like domain, which is responsible for sequence‐specific binding of these proteins to DNA. Previous studies have shown that the absence or non‐functionality of either of TRF proteins results in increased telomere length and chromosomal fusions that ultimately leads to cell death. In this study, we wanted to clarify whether compound C‐1305 and its structural analogs bind telomeric DNA and stabilize G‐quadruplexes. The binding affinities of studied compounds to telomeric DNA were tested against different DNA substrates using microdialysis and DNA melting temperature assays and stabilization of G‐quadruplexes by FRET assay. Given that telomeric repeats contain GGG triplets, we also wanted to assess whether C‐1305 induces structural distortions within telomeric sequences and whether drug‐DNA binding changes the affinity of TRF1 and TRF2 to telomeric DNA. We show here that from all studied compounds C‐1305 showed the highest affinity and specificity to double‐stranded oligonucleotides containing telomeric TTAGGG repeats and to a oligonucleotide folded into intramolecular G‐quadruplex structure. Based on the molecular modeling studies, we also propose a model of C‐1305/G‐quadruplex complex. We recently postulated, that TRF1/2‐telomeric DNA complexes may be excellent targets for anticancer drugs, which by specific binding to telomeric DNA disturb the formation of Shelterin‐DNA complexes. In the second part of this work, we characterized interactions of recombinant TRF1 and TRF2 proteins and Myb‐like domain fragments, with telomeric DNA, in the absence or presence of compound C‐1305 and its analogs. Using chemical probing, we found that C‐1305 induces structural changes within GGG triplets present in the telomeric sequences in double‐stranded DNA substrate. We also showed by the EMSA assay that compound C‐1305 is disturbs the binding of TRF1/2 proteins and Myb‐domain peptide with telomeric DNA probes, containing consensus binding sequences for both studied Shelterins. Together, our results show that compound C‐1305 specifically binds both G‐quadruplex and double‐stranded telomeric DNA. Moreover, this compound induces structural changes in double stranded telomeric DNA, specifically within GGG triplets, that leads to greatly decreased affinity of both TRF1 and TRF2 to telomeric DNA substrates. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C159.
Titia De Lange - One of the best experts on this subject based on the ideXlab platform.
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protection of telomeres 1 proteins pot1a and pot1b can repress atr signaling by rpa exclusion but binding to cst limits atr repression by pot1b
Journal of Biological Chemistry, 2018Co-Authors: Katja Kratz, Titia De LangeAbstract:Comprised of telomeric TTAGGG repeats and Shelterin, telomeres ensure that the natural ends of chromosomes remain impervious to the DNA damage response. Telomeres carry a long constitutive 3' overhang that can bind replication protein A (RPA) and activate the ATR Ser/Thr kinase (ATR), which induces cell cycle arrest. A single-stranded (ss) TTAGGG repeat-binding protein in mouse Shelterin, POT1a, has been proposed to repress ATR signaling by preventing RPA binding. Repression of ATR at telomeres requires tethering of POT1a to the other Shelterin subunits situated on the double-stranded (ds) telomeric DNA. The simplest model of ATR repression, the "tethered exclusion model," suggests that the only critical features of POT1a are its connection to Shelterin and its binding to ss telomeric DNA. In agreement with the model, we show here that a Shelterin-tethered variant of RPA70 (lacking the ATR recruitment domain) can repress ATR signaling at telomeres that lack POT1a. However, arguing against the tethered exclusion model, the nearly identical POT1b subunit of Shelterin has been shown to be much less proficient than POT1a in repression of ATR. We now show that POT1b has the intrinsic ability to fully repress ATR but is prevented from doing so when bound to Ctc1, Stn1, Ten1 (CST), the complex needed for telomere end processing. These results establish that Shelterin represses ATR with a tethered ssDNA-binding domain that excludes RPA from the 3' overhang and also reveal an unexpected effect of CST on the ability of POT1b to repress ATR.
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Shelterin-Mediated Telomere Protection
Annual review of genetics, 2018Co-Authors: Titia De LangeAbstract:For more than a decade, it has been known that mammalian cells use Shelterin to protect chromosome ends. Much progress has been made on the mechanism by which Shelterin prevents telomeres from inadvertently activating DNA damage signaling and double-strand break (DSB) repair pathways. Shelterin averts activation of three DNA damage response enzymes [the ataxia-telangiectasia-mutated (ATM) and ataxia telangiectasia and Rad3-related (ATR) kinases and poly(ADP-ribose) polymerase 1 (PARP1)], blocks three DSB repair pathways [classical nonhomologous end joining (c-NHEJ), alternative (alt)-NHEJ, and homology-directed repair (HDR)], and prevents hyper-resection at telomeres. For several of these functions, mechanistic insights have emerged. In addition, much has been learned about how Shelterin maintains the telomeric 3' overhang, forms and protects the t-loop structure, and promotes replication through telomeres. These studies revealed that Shelterin is compartmentalized, with individual subunits dedicated to distinct aspects of the end-protection problem. This review focuses on the current knowledge of Shelterin-mediated telomere protection, highlights differences between human and mouse Shelterin, and discusses some of the questions that remain.
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What I got wrong about Shelterin.
The Journal of biological chemistry, 2018Co-Authors: Titia De LangeAbstract:The ASBMB 2018 Bert and Natalie Vallee award in Biomedical Sciences honors our work on Shelterin, a protein complex that helps cells distinguish the chromosome ends from sites of DNA damage. Shelterin protects telomeres from all aspects of the DNA damage response, including ATM and ATR serine/threonine kinase signaling and several forms of double-strand break repair. Today, this six-subunit protein complex could easily be identified in one single proteomics step. But, it took us more than 15 years to piece together the entire Shelterin complex, one protein at a time. Although we did a lot of things right, here I tell the story of Shelterin's discovery with an emphasis on the things that I got wrong along the way.
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The DDR at telomeres lacking intact Shelterin does not require substantial chromatin decompaction.
Genes & development, 2017Co-Authors: Leonid A. Timashev, Hazen P. Babcock, Xiaowei Zhuang, Titia De LangeAbstract:Telomeres are protected by Shelterin, a six-subunit protein complex that represses the DNA damage response (DDR) at chromosome ends. Extensive data suggest that TRF2 in Shelterin remodels telomeres into the t-loop structure, thereby hiding telomere ends from double-stranded break repair and ATM signaling, whereas POT1 represses ATR signaling by excluding RPA. An alternative protection mechanism was suggested recently by which Shelterin subunits TRF1, TRF2, and TIN2 mediate telomeric chromatin compaction, which was proposed to minimize access of DDR factors. We performed superresolution imaging of telomeres in mouse cells after conditional deletion of TRF1, TRF2, or both, the latter of which results in the complete loss of Shelterin. Upon removal of TRF1 or TRF2, we observed only minor changes in the telomere volume in most of our experiments. Upon codeletion of TRF1 and TRF2, the telomere volume increased by varying amounts, but even those samples exhibiting small changes in telomere volume showed DDR at nearly all telomeres. Upon Shelterin removal, telomeres underwent 53BP1-dependent clustering, potentially explaining at least in part the apparent increase in telomere volume. Furthermore, chromatin accessibility, as determined by ATAC-seq (assay for transposase-accessible chromatin [ATAC] with high-throughput sequencing), was not substantially altered by Shelterin removal. These results suggest that the DDR induced by Shelterin removal does not require substantial telomere decompaction.
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Telomere Recognition and Assembly Mechanism of Mammalian Shelterin
Cell reports, 2017Co-Authors: Fabian Erdel, Katja Kratz, Smaranda Willcox, Jack D. Griffith, Eric C. Greene, Titia De LangeAbstract:Shelterin is a six-subunit protein complex that plays crucial roles in telomere length regulation, protection, and maintenance. Although several Shelterin subunits have been studied in vitro, the biochemical properties of the fully assembled Shelterin complex are not well defined. Here, we characterize Shelterin using ensemble biochemical methods, electron microscopy, and single-molecule imaging to determine how Shelterin recognizes and assembles onto telomeric repeats. We show that Shelterin complexes can exist in solution and primarily locate telomeric DNA through a three-dimensional diffusive search. Shelterin can diffuse along non-telomeric DNA but is impeded by nucleosomes, arguing against extensive one-dimensional diffusion as a viable assembly mechanism. Our work supports a model in which individual Shelterin complexes rapidly bind to telomeric repeats as independent functional units, which do not alter the DNA-binding mode of neighboring complexes but, rather, occupy telomeric DNA in a "beads on a string" configuration.
Joanna Bidzinska - One of the best experts on this subject based on the ideXlab platform.
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Novel anticancer strategy aimed at targeting Shelterin complexes by the induction of structural changes in telomeric DNA: hitting two birds with one stone.
Current cancer drug targets, 2014Co-Authors: Joanna Bidzinska, Maciej Baginski, Andrzej SkladanowskiAbstract:The ends of chromosomes in mammals are composed of telomeric DNA containing TTAGGG repeats, which bind specific proteins called Shelterins. This telomeric DNA together with Shelterins form a cap that protects the ends of chromosomes from being recognized as sites of DNA damage and from chromosomal fusions. Many very successful antitumor drugs used in the treatment of cancer patients bind to DNA, some of them with a prominent sequence specificity leads to changes in DNA structure and integrity. We propose a new target for antitumor drugs where small molecule ligands can bind to telomeric DNA and induce specific structural changes. These changes would lead to a selective interference with the formation of telomeric DNA-Shelterin complexes, especially involving TRF1 and TRF2 proteins, as these proteins bind double-stranded telomeric DNA in a sequence- and structure-dependent manner. The rationale of the proposed therapeutic strategy is further justified by the fact that tumor cells have relatively short telomeres and frequently de-regulated Shelterin expression and/or functionality. Thus uncapping of chromosome ends by DNA binding compounds which disrupt DNA-Shelterin complexes can ultimately induce selective cytotoxic effect in tumor cells. Possible implications for rational design of new antitumor drugs which interfere with telomeric DNA structure and formation of DNA-Shelterin complexes are discussed.
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Abstract C159: Specific structural changes in telomeric DNA induced by triazoloacridone compound C‐1305 prevent the formation of TRF1/2 Shelterin complexes
Molecular Cancer Therapeutics, 2009Co-Authors: Joanna Bidzinska, Maciej Baginski, Marcin Wojciechowski, Andrzej SkladanowskiAbstract:C‐1305 is a triazoloacridone derivative with potent activity in lung and colon cancer models. We reported previously that compound C‐1305 binds to DNA by intercalation and induces structural distortions in DNA regions containing guanine triplets GGG, at concentrations as low as 0.1 µM. This is a unique property of C‐1305 since similar results were not observed for any of the structurally related triazoloacridone derivatives tested or other DNA binding compounds. Human telomeres consist of tandem repeats of the TTAGGG fragments and form specialized DNA‐protein complexes consisting of telomere binding proteins (Shelterins), localized at the ends of the linear chromosomes. Most of telomeric DNA is double‐stranded except the most extreme part where 3′ region of the G strand is single‐stranded and may form the so‐called G‐quadruplex structures. Two telomere‐binding proteins, TRF1 and TRF2, are the key components of the Shelterin complex that bind double‐stranded telomeric DNA. Both TRF1 and TRF2 contain the so‐called Myb‐like domain, which is responsible for sequence‐specific binding of these proteins to DNA. Previous studies have shown that the absence or non‐functionality of either of TRF proteins results in increased telomere length and chromosomal fusions that ultimately leads to cell death. In this study, we wanted to clarify whether compound C‐1305 and its structural analogs bind telomeric DNA and stabilize G‐quadruplexes. The binding affinities of studied compounds to telomeric DNA were tested against different DNA substrates using microdialysis and DNA melting temperature assays and stabilization of G‐quadruplexes by FRET assay. Given that telomeric repeats contain GGG triplets, we also wanted to assess whether C‐1305 induces structural distortions within telomeric sequences and whether drug‐DNA binding changes the affinity of TRF1 and TRF2 to telomeric DNA. We show here that from all studied compounds C‐1305 showed the highest affinity and specificity to double‐stranded oligonucleotides containing telomeric TTAGGG repeats and to a oligonucleotide folded into intramolecular G‐quadruplex structure. Based on the molecular modeling studies, we also propose a model of C‐1305/G‐quadruplex complex. We recently postulated, that TRF1/2‐telomeric DNA complexes may be excellent targets for anticancer drugs, which by specific binding to telomeric DNA disturb the formation of Shelterin‐DNA complexes. In the second part of this work, we characterized interactions of recombinant TRF1 and TRF2 proteins and Myb‐like domain fragments, with telomeric DNA, in the absence or presence of compound C‐1305 and its analogs. Using chemical probing, we found that C‐1305 induces structural changes within GGG triplets present in the telomeric sequences in double‐stranded DNA substrate. We also showed by the EMSA assay that compound C‐1305 is disturbs the binding of TRF1/2 proteins and Myb‐domain peptide with telomeric DNA probes, containing consensus binding sequences for both studied Shelterins. Together, our results show that compound C‐1305 specifically binds both G‐quadruplex and double‐stranded telomeric DNA. Moreover, this compound induces structural changes in double stranded telomeric DNA, specifically within GGG triplets, that leads to greatly decreased affinity of both TRF1 and TRF2 to telomeric DNA substrates. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C159.
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abstract c159 specific structural changes in telomeric dna induced by triazoloacridone compound c 1305 prevent the formation of trf1 2 Shelterin complexes
Molecular Cancer Therapeutics, 2009Co-Authors: Joanna Bidzinska, Maciej Baginski, Marcin Wojciechowski, Andrzej SkladanowskiAbstract:C‐1305 is a triazoloacridone derivative with potent activity in lung and colon cancer models. We reported previously that compound C‐1305 binds to DNA by intercalation and induces structural distortions in DNA regions containing guanine triplets GGG, at concentrations as low as 0.1 µM. This is a unique property of C‐1305 since similar results were not observed for any of the structurally related triazoloacridone derivatives tested or other DNA binding compounds. Human telomeres consist of tandem repeats of the TTAGGG fragments and form specialized DNA‐protein complexes consisting of telomere binding proteins (Shelterins), localized at the ends of the linear chromosomes. Most of telomeric DNA is double‐stranded except the most extreme part where 3′ region of the G strand is single‐stranded and may form the so‐called G‐quadruplex structures. Two telomere‐binding proteins, TRF1 and TRF2, are the key components of the Shelterin complex that bind double‐stranded telomeric DNA. Both TRF1 and TRF2 contain the so‐called Myb‐like domain, which is responsible for sequence‐specific binding of these proteins to DNA. Previous studies have shown that the absence or non‐functionality of either of TRF proteins results in increased telomere length and chromosomal fusions that ultimately leads to cell death. In this study, we wanted to clarify whether compound C‐1305 and its structural analogs bind telomeric DNA and stabilize G‐quadruplexes. The binding affinities of studied compounds to telomeric DNA were tested against different DNA substrates using microdialysis and DNA melting temperature assays and stabilization of G‐quadruplexes by FRET assay. Given that telomeric repeats contain GGG triplets, we also wanted to assess whether C‐1305 induces structural distortions within telomeric sequences and whether drug‐DNA binding changes the affinity of TRF1 and TRF2 to telomeric DNA. We show here that from all studied compounds C‐1305 showed the highest affinity and specificity to double‐stranded oligonucleotides containing telomeric TTAGGG repeats and to a oligonucleotide folded into intramolecular G‐quadruplex structure. Based on the molecular modeling studies, we also propose a model of C‐1305/G‐quadruplex complex. We recently postulated, that TRF1/2‐telomeric DNA complexes may be excellent targets for anticancer drugs, which by specific binding to telomeric DNA disturb the formation of Shelterin‐DNA complexes. In the second part of this work, we characterized interactions of recombinant TRF1 and TRF2 proteins and Myb‐like domain fragments, with telomeric DNA, in the absence or presence of compound C‐1305 and its analogs. Using chemical probing, we found that C‐1305 induces structural changes within GGG triplets present in the telomeric sequences in double‐stranded DNA substrate. We also showed by the EMSA assay that compound C‐1305 is disturbs the binding of TRF1/2 proteins and Myb‐domain peptide with telomeric DNA probes, containing consensus binding sequences for both studied Shelterins. Together, our results show that compound C‐1305 specifically binds both G‐quadruplex and double‐stranded telomeric DNA. Moreover, this compound induces structural changes in double stranded telomeric DNA, specifically within GGG triplets, that leads to greatly decreased affinity of both TRF1 and TRF2 to telomeric DNA substrates. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C159.
Maciej Baginski - One of the best experts on this subject based on the ideXlab platform.
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Novel anticancer strategy aimed at targeting Shelterin complexes by the induction of structural changes in telomeric DNA: hitting two birds with one stone.
Current cancer drug targets, 2014Co-Authors: Joanna Bidzinska, Maciej Baginski, Andrzej SkladanowskiAbstract:The ends of chromosomes in mammals are composed of telomeric DNA containing TTAGGG repeats, which bind specific proteins called Shelterins. This telomeric DNA together with Shelterins form a cap that protects the ends of chromosomes from being recognized as sites of DNA damage and from chromosomal fusions. Many very successful antitumor drugs used in the treatment of cancer patients bind to DNA, some of them with a prominent sequence specificity leads to changes in DNA structure and integrity. We propose a new target for antitumor drugs where small molecule ligands can bind to telomeric DNA and induce specific structural changes. These changes would lead to a selective interference with the formation of telomeric DNA-Shelterin complexes, especially involving TRF1 and TRF2 proteins, as these proteins bind double-stranded telomeric DNA in a sequence- and structure-dependent manner. The rationale of the proposed therapeutic strategy is further justified by the fact that tumor cells have relatively short telomeres and frequently de-regulated Shelterin expression and/or functionality. Thus uncapping of chromosome ends by DNA binding compounds which disrupt DNA-Shelterin complexes can ultimately induce selective cytotoxic effect in tumor cells. Possible implications for rational design of new antitumor drugs which interfere with telomeric DNA structure and formation of DNA-Shelterin complexes are discussed.
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Abstract C159: Specific structural changes in telomeric DNA induced by triazoloacridone compound C‐1305 prevent the formation of TRF1/2 Shelterin complexes
Molecular Cancer Therapeutics, 2009Co-Authors: Joanna Bidzinska, Maciej Baginski, Marcin Wojciechowski, Andrzej SkladanowskiAbstract:C‐1305 is a triazoloacridone derivative with potent activity in lung and colon cancer models. We reported previously that compound C‐1305 binds to DNA by intercalation and induces structural distortions in DNA regions containing guanine triplets GGG, at concentrations as low as 0.1 µM. This is a unique property of C‐1305 since similar results were not observed for any of the structurally related triazoloacridone derivatives tested or other DNA binding compounds. Human telomeres consist of tandem repeats of the TTAGGG fragments and form specialized DNA‐protein complexes consisting of telomere binding proteins (Shelterins), localized at the ends of the linear chromosomes. Most of telomeric DNA is double‐stranded except the most extreme part where 3′ region of the G strand is single‐stranded and may form the so‐called G‐quadruplex structures. Two telomere‐binding proteins, TRF1 and TRF2, are the key components of the Shelterin complex that bind double‐stranded telomeric DNA. Both TRF1 and TRF2 contain the so‐called Myb‐like domain, which is responsible for sequence‐specific binding of these proteins to DNA. Previous studies have shown that the absence or non‐functionality of either of TRF proteins results in increased telomere length and chromosomal fusions that ultimately leads to cell death. In this study, we wanted to clarify whether compound C‐1305 and its structural analogs bind telomeric DNA and stabilize G‐quadruplexes. The binding affinities of studied compounds to telomeric DNA were tested against different DNA substrates using microdialysis and DNA melting temperature assays and stabilization of G‐quadruplexes by FRET assay. Given that telomeric repeats contain GGG triplets, we also wanted to assess whether C‐1305 induces structural distortions within telomeric sequences and whether drug‐DNA binding changes the affinity of TRF1 and TRF2 to telomeric DNA. We show here that from all studied compounds C‐1305 showed the highest affinity and specificity to double‐stranded oligonucleotides containing telomeric TTAGGG repeats and to a oligonucleotide folded into intramolecular G‐quadruplex structure. Based on the molecular modeling studies, we also propose a model of C‐1305/G‐quadruplex complex. We recently postulated, that TRF1/2‐telomeric DNA complexes may be excellent targets for anticancer drugs, which by specific binding to telomeric DNA disturb the formation of Shelterin‐DNA complexes. In the second part of this work, we characterized interactions of recombinant TRF1 and TRF2 proteins and Myb‐like domain fragments, with telomeric DNA, in the absence or presence of compound C‐1305 and its analogs. Using chemical probing, we found that C‐1305 induces structural changes within GGG triplets present in the telomeric sequences in double‐stranded DNA substrate. We also showed by the EMSA assay that compound C‐1305 is disturbs the binding of TRF1/2 proteins and Myb‐domain peptide with telomeric DNA probes, containing consensus binding sequences for both studied Shelterins. Together, our results show that compound C‐1305 specifically binds both G‐quadruplex and double‐stranded telomeric DNA. Moreover, this compound induces structural changes in double stranded telomeric DNA, specifically within GGG triplets, that leads to greatly decreased affinity of both TRF1 and TRF2 to telomeric DNA substrates. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C159.
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abstract c159 specific structural changes in telomeric dna induced by triazoloacridone compound c 1305 prevent the formation of trf1 2 Shelterin complexes
Molecular Cancer Therapeutics, 2009Co-Authors: Joanna Bidzinska, Maciej Baginski, Marcin Wojciechowski, Andrzej SkladanowskiAbstract:C‐1305 is a triazoloacridone derivative with potent activity in lung and colon cancer models. We reported previously that compound C‐1305 binds to DNA by intercalation and induces structural distortions in DNA regions containing guanine triplets GGG, at concentrations as low as 0.1 µM. This is a unique property of C‐1305 since similar results were not observed for any of the structurally related triazoloacridone derivatives tested or other DNA binding compounds. Human telomeres consist of tandem repeats of the TTAGGG fragments and form specialized DNA‐protein complexes consisting of telomere binding proteins (Shelterins), localized at the ends of the linear chromosomes. Most of telomeric DNA is double‐stranded except the most extreme part where 3′ region of the G strand is single‐stranded and may form the so‐called G‐quadruplex structures. Two telomere‐binding proteins, TRF1 and TRF2, are the key components of the Shelterin complex that bind double‐stranded telomeric DNA. Both TRF1 and TRF2 contain the so‐called Myb‐like domain, which is responsible for sequence‐specific binding of these proteins to DNA. Previous studies have shown that the absence or non‐functionality of either of TRF proteins results in increased telomere length and chromosomal fusions that ultimately leads to cell death. In this study, we wanted to clarify whether compound C‐1305 and its structural analogs bind telomeric DNA and stabilize G‐quadruplexes. The binding affinities of studied compounds to telomeric DNA were tested against different DNA substrates using microdialysis and DNA melting temperature assays and stabilization of G‐quadruplexes by FRET assay. Given that telomeric repeats contain GGG triplets, we also wanted to assess whether C‐1305 induces structural distortions within telomeric sequences and whether drug‐DNA binding changes the affinity of TRF1 and TRF2 to telomeric DNA. We show here that from all studied compounds C‐1305 showed the highest affinity and specificity to double‐stranded oligonucleotides containing telomeric TTAGGG repeats and to a oligonucleotide folded into intramolecular G‐quadruplex structure. Based on the molecular modeling studies, we also propose a model of C‐1305/G‐quadruplex complex. We recently postulated, that TRF1/2‐telomeric DNA complexes may be excellent targets for anticancer drugs, which by specific binding to telomeric DNA disturb the formation of Shelterin‐DNA complexes. In the second part of this work, we characterized interactions of recombinant TRF1 and TRF2 proteins and Myb‐like domain fragments, with telomeric DNA, in the absence or presence of compound C‐1305 and its analogs. Using chemical probing, we found that C‐1305 induces structural changes within GGG triplets present in the telomeric sequences in double‐stranded DNA substrate. We also showed by the EMSA assay that compound C‐1305 is disturbs the binding of TRF1/2 proteins and Myb‐domain peptide with telomeric DNA probes, containing consensus binding sequences for both studied Shelterins. Together, our results show that compound C‐1305 specifically binds both G‐quadruplex and double‐stranded telomeric DNA. Moreover, this compound induces structural changes in double stranded telomeric DNA, specifically within GGG triplets, that leads to greatly decreased affinity of both TRF1 and TRF2 to telomeric DNA substrates. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C159.
Feng Qiao - One of the best experts on this subject based on the ideXlab platform.
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The cooperative assembly of Shelterin bridge provides a kinetic gateway that controls telomere length homeostasis
2020Co-Authors: Jinqiang Liu, Kehan Bao, Jin-kwang Kim, Songtao Jia, Feng QiaoAbstract:Shelterin is a six-proteins complex that coats chromosome ends to ensure their proper protection and maintenance. Similar to the human Shelterin, fission yeast Shelterin is composed of telomeric double- and single-stranded DNA-binding proteins, Taz1 and Pot1, respectively, bridged by Rap1, Poz1, and Tpz1. The assembly of the proteinaceous Tpz1-Poz1-Rap1 complex occurs cooperatively and disruption of this Shelterin bridge leads to unregulated telomere elongation. However, how this biophysical property of bridge assembly is integrated into Shelterin function is not known. Here, utilizing synthetic bridges with a range of binding properties, we find that synthetic Shelterin bridge lacking cooperativity requires a linker pair that matches the native bridge in complex lifespan but has dramatically higher affinity. We find that cooperative assembly confers kinetic properties on the Shelterin bridge allowing disassembly to function as a molecular timer, regulating the duration of the telomere open state, and consequently telomere lengthening to achieve a defined species-specific length range. ### Competing Interest Statement The authors have declared no competing interest.
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Structural Basis for Shelterin Bridge Assembly
Molecular cell, 2017Co-Authors: Jin-kwang Kim, Jinqiang Liu, Kyle W. Roskamp, Banumathi Sankaran, Lan Huang, Elizabeth A. Komives, Feng QiaoAbstract:Telomere elongation through telomerase enables chromosome survival during cellular proliferation. The conserved multifunctional Shelterin complex associates with telomeres to coordinate multiple telomere activities, including telomere elongation by telomerase. Similar to the human Shelterin, fission yeast Shelterin is composed of telomeric sequence-specific double- and single-stranded DNA-binding proteins, Taz1 and Pot1, respectively, bridged by Rap1, Poz1, and Tpz1. Here, we report the crystal structure of the fission yeast Tpz1475-508-Poz1-Rap1467-496 complex that provides the structural basis for Shelterin bridge assembly. Biochemical analyses reveal that Shelterin bridge assembly is a hierarchical process in which Tpz1 binding to Poz1 elicits structural changes in Poz1, allosterically promoting Rap1 binding to Poz1. Perturbation of the cooperative Tpz1-Poz1-Rap1 assembly through mutation of the "conformational trigger" in Poz1 leads to unregulated telomere lengthening. Furthermore, we find that the human Shelterin counterparts TPP1-TIN2-TRF2 also assemble hierarchically, indicating cooperativity as a conserved driving force for Shelterin assembly.
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Spatial Organization and Molecular Interactions of the Schizosaccharomyces pombe Ccq1-Tpz1-Poz1 Shelterin Complex.
Journal of molecular biology, 2017Co-Authors: Harry Scott, Jin-kwang Kim, Feng Qiao, Lan Huang, Derek J. TaylorAbstract:The Shelterin complex is a macromolecular assembly of proteins that binds to and protects telomeric DNA, which composes the ends of all linear chromosomes. Shelterin proteins prevent chromosome ends from fusing together and from eliciting erroneous induction of DNA damage response pathways. In addition, Shelterin proteins play key roles in regulating the recruitment and activation of telomerase, an enzyme that extends telomeric DNA. In fission yeast, Schizosaccharomyces pombe, interactions between the Shelterin proteins Ccq1, Tpz1, and Poz1 are important for regulating telomerase-mediated telomere synthesis and thus telomere length homeostasis. Here, we used electron microscopy combined with genetic labeling to define the three-dimensional arrangement of the S. pombe Ccq1-Tpz1-Poz1 (CTP) complex. Crosslinking mass spectrometry was used to identify individual residues that are in proximity to the protein-protein interfaces of the assembled CTP complex. Together, our data provide a first glimpse into the architectural design of the CTP complex and reveals unique interactions that are important in maintaining the S. pombe telomere in a non-extendible state.
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The proper connection between Shelterin components is required for telomeric heterochromatin assembly
Genes & development, 2016Co-Authors: Jiyong Wang, Jinqiang Liu, Feng Qiao, Allison Cohen, Anudari Letian, Xavier Tadeo, James J. Moresco, John R. Yates, Songtao JiaAbstract:Telomeric regions contain prominent sites of heterochromatin, which is associated with unique histone modification profiles such as the methylation of histone H3 at Lys9 (H3K9me). In fission yeast, the conserved telomeric Shelterin complex recruits the histone H3K9 methyltransferase complex CLRC to establish subtelomeric heterochromatin. Although many Shelterin mutations affect subtelomeric heterochromatin assembly, the mechanism remains elusive due to the diverse functions of Shelterin. Through affinity purification, we found that Shelterin directly associates with CLRC through the Ccq1 subunit. Surprisingly, mutations that disrupt interactions between Shelterin subunits compromise subtelomeric heterochromatin without affecting CLRC interaction with Shelterin component Pot1, located at chromosome ends. We further discovered that telomeric repeats are refractory to heterochromatin spreading and that artificial restoration of Shelterin connections or increased heterochromatin spreading rescued heterochromatin defects in these Shelterin mutants. Thus, subtelomeric heterochromatin assembly requires both the recruitment of CLRC by Shelterin to chromosome ends and the proper connection of Shelterin components, which allows CLRC to skip telomeric repeats to internal regions.
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Dissecting Fission Yeast Shelterin Interactions via MICro-MS Links Disruption of Shelterin Bridge to Tumorigenesis
Cell reports, 2015Co-Authors: Jinqiang Liu, Hyun-ik Jun, Jin-kwang Kim, Lan Huang, Jan C. Bierma, Scott D. Rychnovsky, Feng QiaoAbstract:Shelterin, a six-member complex, protects telomeres from nucleolytic attack and regulates their elongation by telomerase. Here, we have developed a strategy, called MICro-MS (Mapping Interfaces via Crosslinking-Mass Spectrometry), that combines crosslinking-mass spectrometry and phylogenetic analysis to identify contact sites within the complex. This strategy allowed identification of separation-of-function mutants of fission yeast Ccq1, Poz1, and Pot1 that selectively disrupt their respective interactions with Tpz1. The various telomere dysregulation phenotypes observed in these mutants further emphasize the critical regulatory roles of Tpz1-centered Shelterin interactions in telomere homeostasis. Furthermore, the conservation between fission yeast Tpz1-Pot1 and human TPP1-POT1 interactions led us to map a human melanoma-associated POT1 mutation (A532P) to the TPP1-POT1 interface. Diminished TPP1-POT1 interaction caused by hPOT1-A532P may enable unregulated telomere extension, which, in turn, helps cancer cells to achieve replicative immortality. Therefore, our study reveals a connection between Shelterin connectivity and tumorigenicity.
