Daekyu Sun

Interests

Teaching

My expertise lies in the area of Medicinal Chemistry, Biochemistry, and Cell and Molecular Biology. Thus, I . I am involved in teaching Medicinal Chemistry and Pharmacognogy (PCOL 837B), Chemotherapy of Infectious Disease (PCOL 836A), and Metabolic Basis of Pharmacotherapy (PCOL 832) for PharmD students. For graduate programs, I am teaching Principles in Drug Discovery, Design, and Development (PHSC 670) and Cellular Communications and Signal Transduction (PCOL 630B).

Research

One of my current research interest focuses on targeting the transcription of human oncogenes small molecules. Our early work has shown that G-rich and C-rich strands could form specific G-quadruplex or i-motif structures, respectively, on the polypurine/polypyrimidine tract in the proximal promoters of the human Myc, RET, and VEGF genes. Those observations led us to explore a new therapeutic strategy to blunt the expression of those genes using small molecules (anti-transcription agents) that specifically inhibit the transcriptional activation. Ultimately, we hope to directly evaluate the antitumor effects of these agents in a preclinical model of human cancers. Medullary thyroid carcinoma (link is external) (MTC) represents the most frequent initial diagnosis for multiple endocrine neoplasia type 2 (MEN2) and is the most common cause of death in these syndromes. Activating germline RET mutations are known to play a central role in the development of MEN2 syndromes. Therefore, the RET protooncogene has been proposed to have a significant place in the prevention and treatment of cancers caused by these syndromes. Not surprisingly, recently developed molecular therapeutics that target the RET pathway have shown activity in clinical trials of patients with advanced MTC, a disease for which there has been no effective therapy. Our early work has shown that G-rich and C-rich strands could form specific G-quadruplex or i-motif structures, respectively, on the polypurine/polypyrimidine tract in the proximal promoter of the human RET gene. That observation led us to explore a new therapeutic strategy to blunt the effect of mutations in RET associated with MTC using small molecules (anti-RET agents) that specifically inhibit the transcriptional activation of this gene. Ultimately, we hope to directly evaluate the antitumor effects of anti-RET agents in a preclinical model of human MTC, a tumor type characterized by germline point mutations of the RET proto-oncogene. 2) Targeting Tumor angiogenesis by targeting the transcriptional activation of the VEGF geneIt is widely believed that angiogenesis, the formation of new blood vessels, promotes tumor growth by providing oxygen and nutrients to proliferating cancerous cells. The switch to an angiogenic phenotype in cancer cells is often mediated by increased expression of vascular endothelial growth factor (VEGF), which is a pluripotent cytokinine and angiogenic growth factor and is often transcriptionally activated by the transcription factor HIF-1a under hypoxic condition. Our early work has shown that the G-rich and C-rich strands could form specific G-quadruplex or i-motif structures, respectively, on the polypurine/polypyrimidine tract in the proximal promoter of these genes. That observation led us to explore a new therapeutic strategy to repress transcriptional activation of human VEGF gene with small molecules capable of binding selectively to non-canonical DNA structures formed within the promoter region of these genes. The results from our research are anticipated to lead to the discovery of novel anti-angiogenic compounds that we could find important applications in the treatment of human cancers. We are also interested in investigating inducible drugresistant mechanisms in human cancer cells to DNA-damaging anticancer drugs, since the acquisition of chemoresistance toward chemotherapy in cancer cells remains one of the principal obstacles to the effective treatment of malignancies. Recently, by combining global transcriptional profiling and bioinformatics, we have deciphered large networked responses to damage caused by the anticancer drug gemcitabine as a model compound in human cancer cells. This large-scale approach particularly contributed to the identification of numerous gene products potentially associated with chemoresistance to anticancer drugs in cancer cells. The functional roles of these genes in the induction of chemoresistance against these anticancer drugs in human cancer cells will be characterized by further functional analysis. Identified genes with a suspected DNA repair function from our proposed study will be of potential importance in uncovering drug resistance mechanisms of cancer cells and in identifying a potential target for therapeutic intervention.

Courses

Scholarly Contributions

Journals/Publications

• Sun, D. (2023). Adefovir Dipivoxil as a Therapeutic Candidate for Medullary Thyroid Carcinoma: Targeting RET and STAT3 Proto-Oncogenes. Cancers (Basel), 15(7), 2163-2185. doi:doi: 10.3390/cancers15072163
• Sun, D., Alqahtani, T., Kumarasamy, V. M., & Huczynski, A. (2020). Salinomycin and its derivatives as potent RET transcriptional inhibitors for the treatment of medullary thyroid carcinoma. International Journal of Oncology, 56(1), 348-358.. doi:10.3892/ijo.2019.4916.
• Sun, D. (2019). Chromatin Immunoprecipitation Assay to Analyze the Effect of G-Quadruplex Interactive Agents on the Binding of RNA Polymerase II and Transcription Factors to a Target Promoter Region.. Methods and Protocols, Methods in Molecular Biology, 2035, 233-242.. doi:10.1007/978-1-4939-9666-7_13
• Sun, D., Onel, B., Wu, G., Lin, C., & Yang, D. (2019). Electrophoretic Mobility Shift Assay and Dimethyl Sulfate Footprinting for Characterization of G-Quadruplexes and G-Quadruplex-Protein Complexes. Methods in Molecular Biology.
• Sun, D., & Kumarasamy, V. (2017). Demonstration of a potent RET transcriptional inhibitor for the treatment of medullary thyroid carcinoma based on an ellipticine derivative. Internal Journal of Oncology, 51, 145. doi:10.3892/ijo.2017.3994
• Sun, D., & White, J. (2017). Potential Mechanism of Chemo-Resistance to Gemcitabine. Novel Approaches in Drug Designing & Development, 2(3), 555588.
• Sun, D., McConnell, N., Xu, Z., Frett, B., Kumarasamy, V., & Li, H. (2017). Synthesis of Constrained Heterocycles Employing Two Post-Ugi Cyclization Methods for Rapid Library Generation with In Cellulo Activity. ChemistrySelect, 2, 11821. doi:10.1002/slct.201702179
• Kumarasamy, V. M., Shin, Y., White, J., & Sun, D. (2015). Selective repression of RET proto-oncogene in medullary thyroid carcinoma by a natural alkaloid berberine. BMC cancer, 15, 599.
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  The gain-of-function mutation of the RET proto-oncogene, which encodes a receptor tyrosine kinase, is strongly associated with the development of several medullary thyroid carcinomas (MTCs). Thus, the RET protein has been explored as an excellent target for progressive and advanced MTC. In this study we have demonstrated a therapeutic strategy for MTC by suppressing the transcription of RET proto-oncogene though the stabilization of G-quadruplex structure formed on the promoter region of this gene using a natural product berberine.
• Sun, D. (2015). Involvement of G-quadruplex structures in regulation of human RET gene expression by small molecules in human medullary thyroid carcinoma TT cells. Oncogene, 1292-9.
• Keerthi, K., Rajapakse, A., Sun, D., & Gates, K. S. (2013). Synthesis and characterization of a small analogue of the anticancer natural product leinamycin. Bioorganic and Medicinal Chemistry, 21(1), 235-241.
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  PMID: 23168080;PMCID: PMC3532941;Abstract: Leinamycin (1) is a Streptomyces-derived natural product that displays nanomolar IC50 values against human cancer cell lines. In the work described here, we report the synthesis and characterization of a small leinamycin analogue 19 that closely resembles the 'upper-right quadrant' of the natural product, consisting of an alicyclic 1,2-dithiolan-3-one 1-oxide heterocycle connected to an alkene by a two-carbon linker. The results indicate that this small analogue contains the core set of functional groups required to enable thiol-triggered generation of both redox active polysulfides and an episulfonium ion intermediate via the complex reaction cascade first seen in the natural product leinamycin. The small leinamycin analogue 19 caused thiol-triggered oxidative DNA strand cleavage in a manner similar to the natural product, but did not alkyate duplex DNA effectively. This highlights the central role of the 18-membered macrocycle of leinamycin in driving efficient DNA alkylation by the natural product. © 2012 Elsevier Ltd. All rights reserved.
• Sinha, P., Shin, Y., Hays, A. M., Gates, K., & Sun, D. (2013). Cellular responses to the DNA damaging natural compound leinamycin. Journal of Cancer Science and Therapy, 5(9).
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  Abstract: Leinamycin is a thiol dependent DNA alkylating agent which shows very potent activity against various human cancer cell lines (IC50 values in the low nanomolar range). This natural compound forms guanine adducts (N7) in DNA which are converted into abasic sites and simultaneously generates Reactive Oxygen Species (ROS), to produce DNA strand breaks in human cancer cells. Our present study shows that leinamycin induces a group of DNA repair and transcription factor genes involved in DNA repair in a MDA-MB-231 human breast cancer cell line, which can mediate chemoresistance to leinamycin. In addition, N-acetylcysteine decreases leinamycin-mediated ROS production while increasing leinamycin mediated apoptotic cell death, without affecting the induction of repair genes. These data indicate that ROS is not a crucial player in leinamycin induced DNA damage and that a precursor of glutathione, N-acetylcysteine, can potentiate leinamycin mediated cytotoxicity by increasing the activation of leinamycin into its DNA reactive form. © 2012 Sinha P, et al.
• Uribe, D. J., Shin, Y. J., Lau, E., Ebbinghaus, S. W., & Sun, D. (2013). Heterogeneous Nuclear Ribonucleoprotein K Binds to the Cytosine-Rich Sequence of the Hypoxia Inducible Factor 1 Alpha Proximal Promoter that forms a Stable i-motif at Neutral pH. Journal of Physical Chemistry & Biophysics, S5, 001. doi:10.4172/2161-0398.S5-001
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  The proximal promoter of the hypoxia inducible factor 1 alpha (HIF-1α) gene contains a poly-purine/poly-pyrimidine (pPu/pPy) tract, which has been shown to affect 90% of its transcriptional control. The guanine-rich sequence of this pPu/pPy tract has been known to be structurally dynamic, easily forming a G-quadruplex structure with a 1:6:1 folding pattern. In present study, we demonstrated that the cytosine-rich (C-rich) sequence within the pPu/pPy tract of the HIF-1α promoter is able to form two major intramolecular i-motif structures with 3:3:3 or 3:4:2 folding patterns near physiological pH using circular dichroism, bromine footprinting and site-directed mutational analysis. These structures are the first known i-motifs that form at neutral pH, with a transitional pH at 6.9, which have been discovered to form within the proximal promoter sequence of oncogenes. Additionally, electrophoretic mobility shift assays (EMSA) combined with bromine footprinting revealed that heterogeneous nuclear ribonucleoprotein K (hnRNP K) is able to bind to the unfolded state of C-rich sequence in a sequence specific manner. Taken together, our results demonstrate that the i-motif structures that form within the C-rich sequence of the HIF-1α promoter can form under physiological conditions and that hnRNP K can bind to this C-rich sequence.
• Sivaramakrishnan, S., Breydo, L., Sun, D., & Gates, K. S. (2012). The macrocycle of leinamycin imparts hydrolytic stability to the thiol-sensing 1,2-dithiolan-3-one 1-oxide unit of the natural product. Bioorganic and Medicinal Chemistry Letters, 22(11), 3791-3794.
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  PMID: 22560586;PMCID: PMC3376404;Abstract: Reaction of cellular thiols with the 1,2-dithiolan-3-one 1-oxide moiety of leinamycin triggers the generation of DNA-damaging reactive intermediates. Studies with small, synthetic analogues of leinamycin reveal that the macrocyclic portion of the natural product imparts remarkable hydrolytic stability to the 1,2-dithiolan-3-one 1-oxide heterocycle without substantially compromising its thiol-sensing property. © 2012 Elsevier Ltd. All rights reserved.
• Sun, D., Viswesh, V., Hays, A. M., Gates, K., & Sun, D. -. (2012). DNA cleavage induced by antitumor antibiotic leinamycin and its biological consequences. Bioorganic & medicinal chemistry, 20(14).
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  The natural product leinamycin has been found to produce abasic sites in duplex DNA through the hydrolysis of the glycosidic bond of guanine residues modified by this drug. In the present study, using a synthetic oligonucleotide duplex, we demonstrate spontaneous DNA strand cleavage at leinamycin-induced abasic sites through a β-elimination reaction. However, methoxyamine modification of leinamycin-induced abasic sites was found to be refractory to the spontaneous β-elimination reaction. Furthermore, this complex was even resistant to the δ-elimination reaction with hot piperidine treatment. Bleomycin and methyl methanesulfonate also induced strand cleavage in a synthetic oligonucleotide duplex even without thermal treatment. However, methoxyamine has a negligible effect on DNA strand cleavage induced by both drugs, suggesting that the mechanism of DNA cleavage induced by leinamycin might be different from those induced by bleomycin or methyl methanesulfonate. In this study, we also assessed the cytotoxicity of leinamycin against a collection of mammalian cell lines defective in various repair pathways. The mammalian cell line defective in the nucleotide excision repair (NER) or base excision repair (BER) pathways was about 3 to 5 times more sensitive to leinamycin as compared to the parental cell line. In contrast, the radiosensitive mutant xrs-5 cell line deficient in V(D)J recombination showed similar sensitivity towards leinamycin compared to the parental cell line. Collectively, our findings suggest that both NER and BER pathways play an important role in the repair of DNA damage caused by leinamycin.
• Sun, D., Guo, K., & Shin, Y. (2011). Evidence of the formation of G-quadruplex structures in the promoter region of the human vascular endothelial growth factor gene. Nucleic Acids Research, 39(4), 1256-1265.
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  PMID: 20959293;PMCID: PMC3045601;Abstract: The polypurine/polypyrimidine (pPu/pPy) tract of the human vascular endothelial growth factor (VEGF) gene is proposed to be structurally dynamic and to have potential to adopt non-B DNA structures. In the present study, we further provide evidence for the existence of the G-quadruplex structure within this tract both in vitro and in vivo using the dimethyl sulfate (DMS) footprinting technique and nucleolin as a structural probe specifically recognizing G-quadruplex structures. We observed that the overall reactivity of the guanine residues within this tract toward DMS was significantly reduced compared with other guanine residues of the flanking regions in both in vitro and in vivo footprinting experiments. We also demonstrated that nucleolin, which is known to bind to G-quadruplex structures, is able to bind specifically to the G-rich sequence of this region in negatively supercoiled DNA. Our chromatin immunoprecipitation analysis further revealed binding of nucleolin to the promoter region of the VEGF gene in vivo. Taken together, our results are in agreement with our hypothesis that secondary DNA structures, such as G-quadruplexes, can be formed in supercoiled duplex DNA and DNA in chromatin in vivo under physiological conditions similar to those formed in single-stranded DNA templates. The Author(s) 2010. Published by Oxford University Press.2010This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2. 5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. © The Author(s) 2010.
• Sun, D., Uribe, D. J., Guo, K., Shin, Y., & Sun, D. -. (2011). Heterogeneous nuclear ribonucleoprotein K and nucleolin as transcriptional activators of the vascular endothelial growth factor promoter through interaction with secondary DNA structures. Biochemistry, 50(18).
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  The human vascular endothelial growth factor (VEGF) promoter contains a polypurine/polypyrimidine (pPu/pPy) tract that is known to play a critical role in its transcriptional regulation. This pPu/pPy tract undergoes a conformational transition between B-DNA, single-stranded DNA, and atypical secondary DNA structures such as G-quadruplexes and i-motifs. We studied the interaction of the cytosine-rich (C-rich) and guanine-rich (G-rich) strands of this tract with transcription factors heterogeneous nuclear ribonucleoprotein (hnRNP) K and nucleolin, respectively, both in vitro and in vivo and their potential role in the transcriptional control of VEGF. Using chromatin immunoprecipitation (ChIP) assay for our in vivo studies and electrophoretic mobility shift assay (EMSA) for our in vitro studies, we demonstrated that both nucleolin and hnRNP K bind selectively to the G- and C-rich sequences, respectively, in the pPu/pPy tract of the VEGF promoter. The small interfering RNA (siRNA)-mediated silencing of either nucleolin or hnRNP K resulted in the down-regulation of basal VEGF gene, suggesting that they act as activators of VEGF transcription. Taken together, the identification of transcription factors that can recognize and bind to atypical DNA structures within the pPu/pPy tract will provide new insight into mechanisms of transcriptional regulation of the VEGF gene.
• Sun, D. (2010). In vitro footprinting of promoter regions within supercoiled plasmid DNA.. Methods in molecular biology (Clifton, N.J.), 613, 223-233.
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  PMID: 19997887;PMCID: PMC2807360;Abstract: Polypurine/polypyrimidine (pPu/pPy) tracts, which exist in the promoter regions of many growth-related genes, have been proposed to be very dynamic in their conformation. In this chapter, we describe a detailed protocol for DNase I and S1 nuclease footprinting experiments with supercoiled plasmid DNA containing the promoter regions to probe whether there are conformational transitions to B-type DNA, melted DNA, and G-quadruplex structures within this tract. This is demonstrated with the proximal promoter region of the human vascular endothelial growth factor (VEGF) gene, which also contains multiple binding sites for Sp1 and Egr-1 transcription factors.
• Sun, D., & Hurley, L. H. (2010). Biochemical techniques for the characterization of G-quadruplex structures: EMSA, DMS footprinting, and DNA polymerase stop assay. Methods in molecular biology (Clifton, N.J.), 608, 65-79.
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  The proximal promoter region of many human growth-related genes contains a polypurine/polypyrimidine tract that serves as multiple binding sites for Sp1 or other transcription factors. These tracts often contain a guanine-rich sequence consisting of four runs of three or more contiguous guanines separated by one or more bases, corresponding to a general motif known for the formation of an intramolecular G-quadruplex. Recent results provide strong evidence that specific G-quadruplex structures form naturally within these polypurine/polypyrimidine tracts in many human promoter regions, raising the possibility that the transcriptional control of these genes can be modulated by G-quadruplex-interactive agents. In this chapter, we describe three general biochemical methodologies, electrophoretic mobility shift assay (EMSA), dimethylsulfate (DMS) footprinting, and the DNA polymerase stop assay, which can be useful for initial characterization of G-quadruplex structures formed by G-rich sequences.
• Sun, D., Melman, G., LeTourneau, N. J., Hays, A. M., & Melman, A. (2010). Synthesis and antiproliferating activity of iron chelators of hydroxyamino-1,3,5-triazine family. Bioorganic and Medicinal Chemistry Letters, 20(2), 458-460.
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  PMID: 20005708;Abstract: We synthesized and evaluated new specific tridentate iron(III) chelators of 2,6-bis[hydroxyamino]-1,3,5-triazine (BHT) family for use in iron deprivation cancer therapy. Physical properties of BHT chelators are easily customizable allowing easy penetration through cellular membranes. Antiproliferative activity of new BHT chelators was studied on MDA-MB-231 and MiaPaCa cells and compared to a clinically available new oral iron chelator, deferasirox (DFX). The antiproliferative activity of new chelators was found to correlate with iron(III) chelation ability and some of analogs showed substantially higher antiproliferative activity than DFX. © 2009 Elsevier Ltd. All rights reserved.
• Sun, D., Viswesh, V., Gates, K., & Sun, D. -. (2010). Characterization of DNA damage induced by a natural product antitumor antibiotic leinamycin in human cancer cells. Chemical research in toxicology, 23(1).
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  Leinamycin is a structurally novel Streptomyces-derived natural product that displays very potent activity against various human cancer cell lines (IC(50) values in the low nanomolar range). Previous in vitro biochemical studies have revealed that leinamycin alkylates DNA, generates apurinic (AP) sites and reactive oxygen species (ROS), and causes DNA strand breaks. However, it is not clear whether these events occur inside cells. In the present study, we have determined the endogenous amount of AP sites and DNA strand breaks in genomic DNA and the amount of oxidative stress in a human pancreatic carcinoma cell line, MiaPaCa, treated with leinamycin by utilizing the aldehyde-reactive probe assay, the comet assay, and fluorescent probes, respectively. We demonstrated that AP sites are formed rapidly following exposure to leinamycin, and the number of AP sites was increased up to seven-fold in a dose-dependent manner. However, only 25-50% of these sites remain 2 h after media containing drug molecules were aspirated and replaced with fresh media. We also observed leinamycin-induced ROS generation and a concomitant increase in apoptosis of MiaPaCa cells. Because both AP sites and ROS have the potential to generate strand breaks in cellular DNA, the comet assay was utilized to detect damage to nuclear DNA in leinamycin-treated MiaPaCa cell cultures. Both alkaline and neutral electrophoretic analysis revealed that leinamycin produces both single- and double-stranded DNA damage in drug-treated cells in a dose-dependent manner. Taken together, the results suggest that rapid conversion of leinamycin-guanine (N7) adducts into AP sites to produce DNA strand breaks, in synergy with leinamycin-derived ROS, accounts for the exceedingly potent biological activity of this natural product.
• Zheng, J., Liu, X., Yuan, Q., Shin, Y., Sun, D., & Yixin, L. u. (2010). Thiol-dependent DNA cleavage by aminomethylated Beaucage's reagent. Organic and Biomolecular Chemistry, 8(6), 1293-1295.
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  PMID: 20204198;Abstract: Aminomethylated Beaucage's reagent 1 was found to be more potent than 3H-1,2-benzodithiol-3-one 1,1-dioxide (Beaucage's reagent) in causing DNA cleavage. The current study demonstrated the importance of the amino functionality in enhancing DNA-cleaving activities, and such findings may facilitate development of novel sulfur-containing DNA-cleaving molecules in cancer therapy. © 2010 The Royal Society of Chemistry.
• Dexheimer, T. S., Carey, S. S., Zuohe, S., Gokhale, V. M., Hu, X., Murata, L. B., Maes, E. M., Weichsel, A., Sun, D., Meuillet, E. J., Montfort, W. R., & Hurley, L. H. (2009). NM23-H2 may play an indirect role in transcriptional activation of c-myc gene expression but does not cleave the nuclease hypersensitive element III(1). Molecular cancer therapeutics, 8(5), 1363-77.
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  The formation of G-quadruplex structures within the nuclease hypersensitive element (NHE) III(1) region of the c-myc promoter and the ability of these structures to repress c-myc transcription have been well established. However, just how these extremely stable DNA secondary structures are transformed to activate c-myc transcription is still unknown. NM23-H2/nucleoside diphosphate kinase B has been recognized as an activator of c-myc transcription via interactions with the NHE III(1) region of the c-myc gene promoter. Through the use of RNA interference, we confirmed the transcriptional regulatory role of NM23-H2. In addition, we find that further purification of NM23-H2 results in loss of the previously identified DNA strand cleavage activity, but retention of its DNA binding activity. NM23-H2 binds to both single-stranded guanine- and cytosine-rich strands of the c-myc NHE III(1) and, to a lesser extent, to a random single-stranded DNA template. However, it does not bind to or cleave the NHE III(1) in duplex form. Significantly, potassium ions and compounds that stabilize the G-quadruplex and i-motif structures have an inhibitory effect on NM23-H2 DNA-binding activity. Mutation of Arg(88) to Ala(88) (R88A) reduced both DNA and nucleotide binding but had minimal effect on the NM23-H2 crystal structure. On the basis of these data and molecular modeling studies, we have proposed a stepwise trapping-out of the NHE III(1) region in a single-stranded form, thus allowing single-stranded transcription factors to bind and activate c-myc transcription. Furthermore, this model provides a rationale for how the stabilization of the G-quadruplex or i-motif structures formed within the c-myc gene promoter region can inhibit NM23-H2 from activating c-myc gene expression.
• Sun, D., & Hurley, L. H. (2009). The importance of negative superhelicity in inducing the formation of G-quadruplex and i-motif structures in the c-Myc promoter: implications for drug targeting and control of gene expression. Journal of medicinal chemistry, 52(9), 2863-74.
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  The importance of DNA supercoiling in transcriptional regulation has been known for many years, and more recently, transcription itself has been shown to be a source of this superhelicity. To mimic the effect of transcriptionally induced negative superhelicity, the G-quadruplex/i-motif-forming region in the c-Myc promoter was incorporated into a supercoiled plasmid. We show, using enzymatic and chemical footprinting, that negative superhelicity facilitates the formation of secondary DNA structures under physiological conditions. Significantly, these structures are not the same as those formed in single-stranded DNA templates. Together with the recently demonstrated role of transcriptionally induced superhelicity in maintaining a mechanosensor mechanism for controlling the firing rate of the c-Myc promoter, we provide a more complete picture of how c-Myc transcription is likely controlled. Last, these physiologically relevant G-quadruplex and i-motif structures, along with the mechanosensor mechanism for control of gene expression, are proposed as novel mechanisms for small molecule targeting of transcriptional control of c-Myc.
• Sun, D., González, V., Guo, K., Hurley, L., & Sun, D. -. (2009). Identification and characterization of nucleolin as a c-myc G-quadruplex-binding protein. The Journal of biological chemistry, 284(35).
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  myc is a proto-oncogene that plays an important role in the promotion of cellular growth and proliferation. Understanding the regulation of c-myc is important in cancer biology, as it is overexpressed in a wide variety of human cancers, including most gynecological, breast, and colon cancers. We previously demonstrated that a guanine-rich region upstream of the P1 promoter of c-myc that controls 85-90% of the transcriptional activation of this gene can form an intramolecular G-quadruplex (G4) that functions as a transcriptional repressor element. In this study, we used an affinity column to purify proteins that selectively bind to the human c-myc G-quadruplex. We found that nucleolin, a multifunctional phosphoprotein, binds in vitro to the c-myc G-quadruplex structure with high affinity and selectivity when compared with other known quadruplex structures. In addition, we demonstrate that upon binding, nucleolin facilitates the formation and increases the stability of the c-myc G-quadruplex structure. Furthermore, we provide evidence that nucleolin overexpression reduces the activity of a c-myc promoter in plasmid presumably by inducing and stabilizing the formation of the c-myc G-quadruplex. Finally, we show that nucleolin binds to the c-myc promoter in HeLa cells, which indicates that this interaction occurs in vivo. In summary, nucleolin may induce c-myc G4 formation in vivo.
• Sun, D., Guo, K., Gokhale, V., Hurley, L. H., & Sun, D. -. (2008). Intramolecularly folded G-quadruplex and i-motif structures in the proximal promoter of the vascular endothelial growth factor gene. Nucleic acids research, 36(14).
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  A polyguanine/polycytosine (polyG/polyC) tract in the proximal promoter of the vascular endothelial growth factor (VEGF) gene is essential for transcriptional activation. The guanine-rich (G-rich) and cytosine-rich (C-rich) strands on this tract are shown to form specific secondary structures, characterized as G-quadruplexes and i-motifs, respectively. Mutational analysis of the G-rich strand combined with dimethyl sulfate (DMS) footprinting, a polymerase stop assay, and circular dichroism (CD) spectroscopy revealed that the G-quadruplex containing a 1:4:1 double-chain reversal loop is the most thermodynamically stable conformation that this strand readily adopts. These studies provide strong evidence that the size of loop regions plays a critical role in determining the most favored folding pattern of a G-quadruplex. The secondary structure formed on the complementary C-rich strand was also determined by mutational analysis combined with Br(2) footprinting and CD spectroscopy. Our results reveal that at a pH of 5.9 this strand is able to form an intramolecular i-motif structure that involves six C-C(+) base pairs and a 2:3:2 loop configuration. Taken together, our results demonstrate that the G-quadruplex and i-motif structures are able to form on the G- and C-rich strands, respectively, of the polyG/polyC tract in the VEGF proximal promoter under conditions that favor the transition from B-DNA to non-B-DNA conformations.
• Sun, D., Liu, W., Guo, K., Rusche, J. J., Ebbinghaus, S., Gokhale, V., & Hurley, L. H. (2008). The proximal promoter region of the human vascular endothelial growth factor gene has a G-quadruplex structure that can be targeted by G-quadruplex-interactive agents. Molecular cancer therapeutics, 7(4), 880-9.
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  Previous studies on the functional analysis of the human vascular endothelial growth factor (VEGF) promoter using the full-length VEGF promoter reporter revealed that the proximal 36-bp region (-85 to -50 relative to transcription initiation site) is essential for basal or inducible VEGF promoter activity in several human cancer cells. This region consists of a polypurine (guanine) tract that contains four runs of at least three contiguous guanines separated by one or more bases, thus conforming to a general motif capable of forming an intramolecular G-quadruplex. Here, we show that the G-rich strand in this region is able to form an intramolecular propeller-type parallel-stranded G-quadruplex structure in vitro by using the electrophoretic mobility shift assay, dimethyl sulfate footprinting technique, the DNA polymerase stop assay, circular dichroism spectroscopy, and computer-aided molecular modeling. Two well-known G-quadruplex-interactive agents, TMPyP4 and Se2SAP, stabilize G-quadruplex structures formed by this sequence in the presence of a potassium ion, although Se2SAP is at least 10-fold more effective in binding to the G-quadruplex than TMPyP4. Between these two agents, Se2SAP better suppresses VEGF transcription in different cancer cell lines, including HEC1A and MDA-MB-231. Collectively, our results provide evidence that specific G-quadruplex structures can be formed in the VEGF promoter region, and that the transcription of this gene can be controlled by ligand-mediated G-quadruplex stabilization. Our results also provide further support for the idea that G-quadruplex structures may play structural roles in vivo and therefore might provide insight into novel methodologies for rational drug design.
• Guo, K., Pourpak, A., Beetz-Rogers, K., Gokhale, V., Sun, D., & Hurley, L. H. (2007). Formation of pseudosymmetrical G-quadruplex and i-motif structures in the proximal promoter region of the RET oncogene. Journal of the American Chemical Society, 129(33), 10220-8.
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  A polypurine (guanine)/polypyrimidine (cytosine)-rich sequence within the proximal promoter region of the human RET oncogene has been shown to be essential for RET basal transcription. Specifically, the G-rich strand within this region consists of five consecutive runs of guanines, which is consistent with the general motif capable of forming intramolecular G-quadruplexes. Here we demonstrate that, in the presence of 100 mM K+, this G-rich strand has the ability to adopt two intramolecular G-quadruplex structures in vitro. Moreover, comparative circular dichroism (CD) and DMS footprinting studies have revealed that the 3'-G-quadruplex structure is a parallel-type intramolecular structure containing three G-tetrads. The G-quadruplex-interactive agents TMPyP4 and telomestatin further stabilize this G-quadruplex structure. In addition, we demonstrate that the complementary C-rich strand forms an i-motif structure in vitro, as shown by CD spectroscopy and chemical footprinting. This 19-mer duplex sequence is predicted to form stable intramolecular G-quadruplex and i-motif species having minimum symmetrical loop sizes of 1:3:1 and 2:3:2, respectively. Together, our results indicate that stable G-quadruplex and i-motif structures can form within the proximal promoter region of the human RET oncogene, suggesting that these secondary structures play an important role in transcriptional regulation of this gene.
• Qin, Y., Rezler, E. M., Gokhale, V., Sun, D., & Hurley, L. H. (2007). Characterization of the G-quadruplexes in the duplex nuclease hypersensitive element of the PDGF-A promoter and modulation of PDGF-A promoter activity by TMPyP4. Nucleic acids research, 35(22), 7698-713.
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  The proximal 5'-flanking region of the human platelet-derived growth factor A (PDGF-A) promoter contains one nuclease hypersensitive element (NHE) that is critical for PDGF-A gene transcription. On the basis of circular dichroism (CD) and electrophoretic mobility shift assay (EMSA), we have shown that the guanine-rich (G-rich) strand of the DNA in this region can form stable intramolecular parallel G-quadruplexes under physiological conditions. A Taq polymerase stop assay has shown that the G-rich strand of the NHE can form two major G-quadruplex structures, which are in dynamic equilibrium and differentially stabilized by three G-quadruplex-interactive drugs. One major parallel G-quadruplex structure of the G-rich strand DNA of NHE was identified by CD and dimethyl sulfate (DMS) footprinting. Surprisingly, CD spectroscopy shows a stable parallel G-quadruplex structure formed within the duplex DNA of the NHE at temperatures up to 100 degrees C. This structure has been characterized by DMS footprinting in the double-stranded DNA of the NHE. In transfection experiments, 10 microM TMPyP4 reduced the activity of the basal promoter of PDGF-A approximately 40%, relative to the control. On the basis of these results, we have established that ligand-mediated stabilization of G-quadruplex structures within the PDGF-A NHE can silence PDGF-A expression.
• Dexheimer, T. S., Sun, D., & Hurley, L. H. (2006). Deconvoluting the structural and drug-recognition complexity of the G-quadruplex-forming region upstream of the bcl-2 P1 promoter. Journal of the American Chemical Society, 128(16), 5404-15.
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  The human bcl-2 gene contains a GC-rich region upstream of the P1 promoter that has been shown to be critically involved in the regulation of bcl-2 gene expression. We have demonstrated that the guanine-rich strand of the DNA in this region can form any one of three distinct intramolecular G-quadruplex structures. Mutation and deletion analysis permitted isolation and identification of three overlapping DNA sequences within this element that formed the three individual G-quadruplexes. Each of these was characterized using nondenaturing gel analysis, DMS footprinting, and circular dichroism. The central G-quadruplex, which is the most stable, forms a mixed parallel/antiparallel structure consisting of three tetrads connected by loops of one, seven, and three bases. Three different G-quadruplex-interactive agents were found to further stabilize these structures, with individual selectivity toward one or more of these G-quadruplexes. Collectively, these results suggest that the multiple G-quadruplexes identified in the promoter region of the bcl-2 gene are likely to play a similar role to the G-quadruplexes in the c-myc promoter in that their formation could serve to modulate gene transcription. Last, we demonstrate that the complexity of the G-quadruplexes in the bcl-2 promoter extends beyond the ability to form any one of three separate G-quadruplexes to each having the capacity to form either three or six different loop isomers. These results are discussed in relation to the biological significance of this G-quadruplex-forming element in modulation of bcl-2 gene expression and the inherent complexity of the system where different G-quadruplexes and loop isomers are possible.
• De Armond, R., Wood, S., Sun, D., Hurley, L. H., & Ebbinghaus, S. W. (2005). Evidence for the presence of a guanine quadruplex forming region within a polypurine tract of the hypoxia inducible factor 1alpha promoter. Biochemistry, 44(49), 16341-50.
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  The promoter of the hypoxia inducible factor 1 alpha (HIF-1alpha) gene has a polypurine/polypyrimidine tract (-65 to -85) overlapping or adjacent to several putative transcription factor binding sites, and we found that mutagenesis of this region diminished basal HIF-1alpha expression. Oligonucleotides representing this region of the HIF-1alpha promoter were analyzed by electrophoretic mobility shift, chemical probing, circular dichroism, and DNA polymerase arrest assays. The guanine-rich strand was found to form a parallel, unimolecular quadruplex in the presence of potassium that was further stabilized by two known quadruplex binding compounds, the cationic porphyrin TmPyP4 and the natural product telomestatin, while TmPyP2, a positional isomer of TmPyP4, did not stabilize quadruplex formation. These data suggest that a quadruplex structure may form in a region of the HIF-1alpha promoter that regulates basal HIF-1alpha expression.
• Liu, W., Sun, D., & Hurley, L. H. (2005). Binding of G-quadruplex-interactive agents to distinct G-quadruplexes induces different biological effects in MiaPaCa cells. Nucleosides, nucleotides & nucleic acids, 24(10-12), 1801-15.
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  Our previous studies have demonstrated the preference of telomestatin for intramolecular, rather than the intermolecular, G-quadruplex structures, while TiMPyP4 has selectivity for intermolecular over intramolecular G-quadruplex structures. However, it was not clear whether the difference in the selectivity between two different G-quadruplex-interactive agents could determine the corresponding biological effects in cultured human tumor cells. Here we evaluated the biological effects of both TMPyP4 and telomestatin in the human pancreatic carcinoma cell line (MiaPaCa) using subtoxic and cytotoxic concentrations. The cytotoxicity of these agents against MiaPaCa cells is quite different, and the IC50 of telomestatin (0.5 microM) is about 100 times less than that of TMPyP4 (50 microM). At IC50 concentrations, TMPyP4 induced anaphase bridge formation in MiaPaCa cells, while telomestatin failed to induce anaphase bridge formation. At subtoxic concentrations, TMPyP4 induced MiaPaCa cell growth arrest, senescence, apoptosis, and telomere length shortening within 5 weeks, while similar biological effects were evident after 12 weeks following treatment with telomestatin. Our data suggest that binding of G-quadruplex-interactive agents to distinct G-quadruplexes could induce different biological effects in human cancer cells.
• Seenisamy, J., Bashyam, S., Gokhale, V., Vankayalapati, H., Sun, D., Siddiqui-Jain, A., Streiner, N., Shin-Ya, K., White, E., Wilson, W. D., & Hurley, L. H. (2005). Design and synthesis of an expanded porphyrin that has selectivity for the c-MYC G-quadruplex structure. Journal of the American Chemical Society, 127(9), 2944-59.
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  Cationic porphyrins are known to bind to and stabilize different types of G-quadruplexes. Recent studies have shown the biological relevance of the intramolecular parallel G-quadruplex as a transcriptional silencer in the c-MYC promoter. TMPyP4 also binds to this G-quadruplex and most likely converts it to a mixed parallel/antiparallel G-quadruplex with two external lateral loops and one internal propeller loop, suppressing c-MYC transcriptional activation. To achieve therapeutic selectivity by targeting G-quadruplexes, it is necessary to synthesize drugs that can differentiate among the different types of G-quadruplexes. We have designed and synthesized a core-modified expanded porphyrin analogue, 5,10,15,20-[tetra(N-methyl-3-pyridyl)]-26,28-diselenasapphyrin chloride (Se2SAP). Se2SAP converts the parallel c-MYC G-quadruplex into a mixed parallel/antiparallel G-quadruplex with one external lateral loop and two internal propeller loops, resulting in strong and selective binding to this G-quadruplex. A Taq polymerase stop assay was used to evaluate the binding of TMPyP4 and Se2SAP to G-quadruplex DNA. Compared to TMPyP4, Se2SAP shows a greater selectivity for and a 40-fold increase in stabilization of the single lateral-loop hybrid. Surface plasmon resonance and competition experiments with duplex DNA and other G-quadruplexes further confirmed the selectivity of Se2SAP for the c-MYC G-quadruplex. Significantly, Se2SAP was found to be less photoactive and noncytotoxic in comparison to TMPyP4. From this study, we have identified an expanded porphyrin that selectively binds with the c-MYC G-quadruplex in the presence of duplex DNA and other G-quadruplexes.
• Sun, D., Guo, K., Rusche, J. J., & Hurley, L. H. (2005). Facilitation of a structural transition in the polypurine/polypyrimidine tract within the proximal promoter region of the human VEGF gene by the presence of potassium and G-quadruplex-interactive agents. Nucleic acids research, 33(18), 6070-80.
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  The proximal promoter region of the human vascular endothelial growth factor (VEGF) gene contains a polypurine/polypyrimidine tract that serves as a multiple binding site for Sp1 and Egr-1 transcription factors. This tract contains a guanine-rich sequence consisting of four runs of three or more contiguous guanines separated by one or more bases, corresponding to a general motif for the formation of an intramolecular G-quadruplex. In this study, we observed the progressive unwinding of the oligomer duplex DNA containing this region into single-stranded forms in the presence of KCl and the G-quadruplex-interactive agents TMPyP4 and telomestatin, suggesting the dynamic nature of this tract under conditions which favor the formation of the G-quadruplex structures. Subsequent footprinting studies with DNase I and S1 nucleases using a supercoiled plasmid DNA containing the human VEGF promoter region also revealed a long protected region, including the guanine-rich sequences, in the presence of KCl and telomestatin. Significantly, a striking hypersensitivity to both nucleases was observed at the 3'-side residue of the predicted G-quadruplex-forming region in the presence of KCl and telomestatin, indicating altered conformation of the human VEGF proximal promoter region surrounding the guanine-rich sequence. In contrast, when specific point mutations were introduced into specific guanine residues within the G-quadruplex-forming region (Sp1 binding sites) to abolish G-quadruplex-forming ability, the reactivity of both nucleases toward the mutated human VEGF proximal promoter region was almost identical, even in the presence of telomestatin with KCl. This comparison of wild-type and mutant sequences strongly suggests that the formation of highly organized secondary structures such as G-quadruplexes within the G-rich region of the human VEGF promoter region is responsible for observed changes in the reactivity of both nucleases within the polypurine/polypyrimidine tract of the human VEGF gene. The formation of the G-quadruplex structures from this G-rich sequence in the human VEGF promoter is further confirmed by the CD experiments. Collectively, our results provide strong evidence that specific G-quadruplex structures can naturally be formed by the G-rich sequence within the polypurine/polypyrimidine tract of the human VEGF promoter region, raising the possibility that the transcriptional control of the VEGF gene can be modulated by G-quadruplex-interactive agents.
• Sun, D., & Urrabaz, R. (2004). Development of non-electrophoretic assay method for DNA ligases and its application to screening of chemical inhibitors of DNA ligase I. Journal of Biochemical and Biophysical Methods, 59(1), 49-59.
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  PMID: 15134906;Abstract: A new rapid assay method for DNA ligases has been developed, which allows direct quantification of enzyme activity without using the traditional polyacrylamide gel electrophoretic technique. In this method, the 5′-biotinylated nicked duplex was used as a substrate for the ligase reaction, in which the 5′-end of the first oligonucleotide (19-mer) on the nicked strand is biotinylated and the second oligonucleotide (20-mer) on the same strand is labeled with radioactive 32P at the 5′-end. After ligation of the biotinylated 19-mer oligonucleotide into the second oligonucleotide with the reaction of DNA ligases, the biotinylated 19-mer oligonucleotide is converted into the radioactive 39-mer oligonucleotide. The ligase reaction products were heat-denatured to release both ligated and unligated biotinylated oligonucleotides. The biotinylated oligonucleotides were then captured on a streptavidin-coated microtiter plate and counted. The results obtained using this method correlated very well with those from the standard assay method using electrophoresis. Using this assay method, we were able to screen a chemical library and identify new DNA ligase inhibitors structurally related to resorcinol, which has growth inhibitory effects on the human breast cancer cell, MCF-7. The method described here is anticipated to be very useful for screening DNA ligase inhibitors from chemical libraries. © 2004 Published by Elsevier B.V.
• Sun, D., Bassett, S., Urrabaz, R., & Sun, D. -. (2004). Cellular response and molecular mechanism of antitumor activity by leinamycin in MiaPaCa human pancreatic cancer cells. Anti-cancer drugs, 15(7).
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  Previous in vitro biochemical studies have revealed that the antitumor drug leinamycin causes oxidative DNA damage and DNA alkylation. However, it is still not clear whether the same mechanism(s) of action operate in cultured human tumor cells. Here, we evaluated the effects of leinamycin in the human pancreatic carcinoma cell line MiaPaCa. Leinamycin was highly toxic to MiaPaCa cells in vitro, with an IC50 value of 50 nM, and extensive DNA fragmentation was observed in leinamycin-treated MiaPaCa cells. Flow cytometric experiments showed that leinamycin was able to disrupt normal cell cycle progression, resulting in an initial arrest of the cells in S phase. With increased time or at higher concentrations of leinamycin, the population of cells in the sub-G1 phase gradually increased, indicative of apoptotic cell death due to DNA damage. Mammalian Chk2, but not Chk1 kinase, was found to be activated in MiaPaCa cells treated with leinamycin, indicating that cellular responses to leinamycin could be attributed to DNA strand break formation rather than DNA adduct formation. Like other DNA-damaging anticancer drugs, the downregulation of telomerase activity was also observed in MiaPaCa cells at cytotoxic concentrations. However, leinamycin failed to induce DNA ligase I expression in MiaPaCa cells, unlike other DNA-damaging agents, which are known to inhibit DNA replication by arresting DNA replication forks. Taken together, the results from our study indicate that the DNA strand breakage caused by the oxidative DNA-damaging property of leinamycin is directly related to the cellular responses of this drug in MiaPaCa cells over the DNA alkylation property in a dose-responsive manner.
• Sun, D. (2002). Biotinylated primer for detecting telomerase activity without amplification.. Methods in molecular biology (Clifton, N.J.), 191, 165-171.
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  PMID: 11951605;
• Sun, D., Urrabaz, R., Buzello, C., & Nguyen, M. (2002). Effects of cisplatin on expression of DNA ligases in MiaPaCa human pancreatic cancer cells. Biochemical and Biophysical Research Communications, 298(4), 537-544.
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  PMID: 12408985;Abstract: The effect of the broad-spectrum anticancer agent, cisplatin, on the expression of DNA ligase I in human pancreatic carcinoma MiaPaCa cells was examined in this study, since DNA ligase I is known to be involved in various DNA repair pathways. Upon exposure of MiaPaCa cells to cisplatin at near IC50 value (2.5-5 μM), about 2-3-fold increase of DNA ligase I levels was observed within 24 h, while levels of other DNA ligases (III and IV) remained unchanged or slightly decreased. The same fold-increase in DNA ligase I levels was also observed in MiaPaCa cells exposed to cytostatic concentrations, but not cytotoxic concentrations of cisplatin, which significantly reduced the number of cells. Flow cytometric analysis revealed that normal cell cycle progression was disrupted in the cells treated with cisplatin, resulting in an initial arrest of the cells in the S-phase, concomitant with a decrease of cells in G0/G1-phase. With time elapsing, the transition from S- to G2 + M-phase was observed, but further progression into G0/G1-phase was blocked. Overall, the increase of DNA ligase I expression seems to correlate well with the arrest of the cell cycle between the S- and G2-phases in response to cisplatin treatment. Interestingly, the cisplatin-induced DNA ligase I increase was abrogated by caffeine treatment in MiaPaCa cells, suggesting that caffeine sensitive kinases might be important mediators in the pathway, leading to the increase of DNA ligase I levels in response to cisplatin. We propose that the increase of DNA ligase I expression after exposure to cisplatin might be required for aiding the cells to recover from the damage by facilitating the repair process. © 2002 Elsevier Science (USA). All rights reserved.
• Sun, D., Urrabaz, R., Buzello, C., & Nguyen, M. (2002). Induction of DNA ligase I by 1-β-D-arabinosylcytosine and aphidicolin in MiaPaCa human pancreatic cancer cells. Experimental Cell Research, 280(1), 90-96.
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  PMID: 12372342;Abstract: Exposure of MiaPaCa cells to 1-β-D-arabinosylcytosine (ara-C) resulted in an increase in DNA ligase levels up to threefold compared to that in the untreated control cells, despite significant growth inhibition. Increased levels of DNA ligase I protein appear to correlate with the appearance of increased mRNA levels. The [3H]thymidine incorporation experiment and the biochemical assay of total polymerase activity revealed that an increase in DNA ligase I levels after treatment with ara-C was not accompanied by an increase of DNA synthesis or an increased presence of DNA polymerase activity inside cells. When cells resumed DNA synthesis after drug treatment, DNA ligase I levels began to drop, indicating that increased DNA ligase I is not required for DNA synthesis. An increase in DNA ligase I was also observed in cells treated with aphidicolin, another inhibitor of DNA synthesis that inhibits DNA polymerases without incorporating itself into DNA, indicating that an increase in DNA ligase I levels could be caused by the arrest of DNA replication by these agents. Interestingly, caffeine, which is a well-known inhibitor of DNA damage checkpoint kinases, abrogated the increase in DNA ligase I in MiaPaCa cells treated with ara-C and aphidicolin, suggesting that caffeine-sensitive kinases might be important mediators in the pathway leading to the increase in DNA ligase I levels in response to anticancer drugs, including ara-C and aphidicolin. We propose that ara-C and aphidicolin induce damage to the DNA strand by arresting DNA replication forks and subsequently increase DNA ligase I levels to facilitate repair of DNA damage. © 2002 Elsevier Science (USA).
• Sun, D., Urrabaz, R., Kelly, S., Nguyen, M., & Weitman, S. (2002). Enhancement of DNA ligase I level by gemcitabine in human cancer cells. Clinical Cancer Research, 8(4), 1189-1195.
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  PMID: 11948132;Abstract: Purpose: DNA ligase I is an essential enzyme for completing DNA replication and DNA repair by ligating Okazaki fragments and by joining single-strand breaks formed either directly by DNA-damaging agents or indirectly by DNA repair enzymes, respectively. In this study, we examined whether the DNA ligase I level could be modulated in human tumor cell lines by treatment with gemcitabine (2′, 2′-difluoro-2′-deoxycytidine), which is a nucleoside analogue of cytidine with proven antitumor activity against a broad spectrum of human cancers in clinical studies. Experimental Design: To determine the effect of gemcitabine on DNA ligase I expression, Western blot analysis was used to measure the DNA ligase I levels in MiaPaCa, NGP, and SK-N-BE cells treated with different concentrations of gemcitabine and harvested at different time intervals. Cell cycle analysis was also performed to determine the underlying mechanism of DNA ligase I level enhancement in response to gemcitabine. In addition, other agents that share the same mechanism of action with gemcitabine were used to elucidate further details. Results: When different types of tumor cell lines, including MiaPaCa, NGP, and SK-N-BE, were treated with gemcitabine, the level of DNA ligase I increased severalfold despite significant cell growth inhibition. In contrast, other DNA ligases (III and IV) either remained unchanged or decreased with treatment. Cell cycle analysis showed that arrest in S-phase corresponded to an increase of DNA ligase I levels in gemcitabine treated cells. Other agents, such as 1-β-D-arabinofuranosylcytosine and hydroxyurea, which partly share mechanisms of action with gemcitabine by targeting DNA polymerases and ribonucleotide reductase, respectively, also caused an increase of DNA ligase I levels. However, 5-fluorouracil, which predominantly targets thymidylate synthase, did not cause an increase of DNA ligase I level. Conclusions: Our results suggest that an arrest of DNA replication caused by gemcitabine treatment through incorporation of gemcitabine triphosphate into replicating DNA and inhibition of ribonucleotide reductase would trigger an increase in DNA ligase I levels in cancer cells. The elevated presence of DNA ligase I in S-phase-arrested cells leads us to speculate that DNA ligase I might have an important role in repairing DNA damage caused by stalled replication forks.
• Duan, W., Rangan, A., Vankayalapati, H., Kim, M. Y., Zeng, Q., Sun, D., Han, H., Fedoroff, O. Y., Nishioka, D., Rha, S. Y., Izbicka, E., Von Hoff, D. D., & Hurley, L. H. (2001). Design and synthesis of fluoroquinophenoxazines that interact with human telomeric G-quadruplexes and their biological effects. Molecular cancer therapeutics, 1(2), 103-20.
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  In this study we have identified a new structural motif for a ligand with G-quadruplex interaction that results in biological effects associated with G-quadruplex-interactive compounds. Fluoroquinolones have been reported to possess weak telomerase inhibitory activity in addition to their better known bacterial gyrase poisoning. Starting with a fluoroquinobenzoxazine, which has modest potency in a human topoisomerase II assay, we have designed a more potent inhibitor of telomerase that has lost its topoisomerase II poisoning activity. This fluoroquinophenoxazine (FQP) interacts with G-quadruplex structures to inhibit the progression of Taq polymerase in a G-quadruplex polymerase stop assay. In addition, we demonstrate by 1H NMR studies that this compound interacts with telomeric G-quadruplex structures by external stacking to the G-tetrad with both the unimolecular fold-over and the parallel G-quadruplex structures. A photocleavage assay confirms the FQP interaction site, which is located off center of the external tetrad but within the loop region. Molecular modeling using simulated annealing was performed on the FQP-parallel G-quadruplex complex to determine the optimum FQP orientation and key molecular interactions with the telomeric G-quadruplex structure. On the basis of the results of these studies, two additional FQP analogues were synthesized, which were designed to test the importance of these key interactions. These analogues were evaluated in the Taq polymerase stop assay for G-quadruplex interaction. The data from this study and the biological evaluation of these three FQPs, using cytotoxicity and a sea urchin embryo system, were in accord with the predicted more potent telomeric G-quadruplex interactions of the initial lead compound and one of the analogues. On the basis of these structural and biological studies, the design of more potent and selective telomeric G-quadruplex-interactive compounds can be envisaged.
• Kerwin, S. M., Sun, D., Kern, J. T., Rangan, A., & Thomas, P. W. (2001). G-quadruplex DNA binding by a series of carbocyanine dyes. Bioorganic and Medicinal Chemistry Letters, 11(18), 2411-2414.
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  PMID: 11549435;Abstract: We have examined a number of carbocyanine dyes for their ability to bind intramolecular G-quadruplex DNA structures (G4′-DNA) using a Taq polymerase stop assay. Of the five dyes examined, only one, N,N'-diethylthiacarbocyanine iodide (DTC), was found to bind to G4′-DNA. DTC was also the only dye found to inhibit human telomerase at 50 μM concentration. © 2001 Elsevier Science Ltd. All rights reserved.
• Shi, D. F., Wheelhouse, R. T., Sun, D., & Hurley, L. H. (2001). Quadruplex-interactive agents as telomerase inhibitors: synthesis of porphyrins and structure-activity relationship for the inhibition of telomerase. Journal of medicinal chemistry, 44(26), 4509-23.
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  The cationic porphyrin 5,10,15,20-tetra-(N-methyl-4-pyridyl)porphyrin (TMPyP4) binds to quadruplex DNA and is thereby an inhibitor of human telomerase (Wheelhouse et al. J. Am. Chem. Soc. 1998, 120, 3261-3262). Herein the synthesis and telomerase-inhibiting activity of a wide range of analogues of TMPyP4 are reported, from which rules for a structure-activity relationship (SAR) have been discerned: (1) stacking interactions are critical for telomerase inhibition, (2) positively charged substituents are important but may be interchanged and combined with hydrogen-bonding groups, and (3) substitution is tolerated only on the meso positions of the porphyrin ring, and the bulk of the substituents should be matched to the width of the grooves in which they putatively lie. This SAR is consistent with a model presented for the complexation of TMPyP4 with human telomeric quadruplex DNA.
• Sun, D., Urrabaz, R., Nguyen, M., Marty, J., Stringer, S., Cruz, E., Medina-Gundrum, L., & Weitman, S. (2001). Elevated expression of DNA ligase I in human cancers. Clinical Cancer Research, 7(12), 4143-4148.
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  PMID: 11751514;Abstract: Purpose: Human DNA ligase I plays an essential role in DNA replication, recombination, and repair by catalyzing the formation of phosphodiester bonds between adjacent 5′-phosphoryl and 3′-hydroxyl termini at single breaks in duplex DNA molecules. DNA ligase I is responsible for the majority of DNA ligase activity present in proliferating cells among four different forms of DNA ligases, designated DNA ligase I, II, III, and IV. In this study, we were interested in comparing DNA ligase I level in human tumors versus normal tissues and in studying whether the inhibition of DNA ligase I could lead to tumor cell death. Experimental Design: DNA ligase I level was measured by Western immunoblot assay in various human malignant tumor specimens and benign tissues obtained from patients, in peripheral blood lymphocytes obtained from healthy donors, and in human tumors grown in nude mice. We also have designed antisense oligonucleotides (ODNs) targeting the mRNA of DNA ligase I and tested whether DNA ligase I antisense ODNs could control tumor cell growth. Results: The amount of DNA ligase I enzyme in malignant tumors was considerably higher than that in benign normal tissues and peripheral blood lymphocytes. The level of DNA ligase I in human tumors grown in nude mice was also very high, and the expression of DNA ligase I appears to be constitutive during in vivo tumor development. We have observed that DNA ligase I antisense ODN inhibited tumor cell growth in a dose-response manner, but nonspecific control ODNs had little effect on the growth of the same cell lines. Conclusions: The presence of DNA ligase I at higher levels in human tumors than in benign normal tissues and normal peripheral lymphocytes suggests DNA ligase I plays more of a role in proliferating cells than in resting cells. These results, together with our finding that DNA ligase I antisense ODNs can suppress tumor cell proliferation, warrant the design and testing of human DNA ligase I inhibitors as new anticancer agents.
• Hurley, L. H., Wheelhouse, R. T., Sun, D., Kerwin, S. M., Salazar, M., Fedoroff, O. Y., Han, F. X., Han, H., Izbicka, E., & Von Hoff, D. D. (2000). G-quadruplexes as targets for drug design. Pharmacology & therapeutics, 85(3), 141-58.
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  G-quadruplexes are a family of secondary DNA structures formed in the presence of monovalent cations that consist of four-stranded structures in which Hoogsteen base-pairing stabilizes G-tetrad structures. These structures are proposed to exist in vivo, although direct confirmatory evidence is lacking. Guanine-rich regions of DNA capable of forming G-quadruplex structures are found in a variety of chromosomal regions, including telomeres and promoter regions of DNA. In this review, we describe the design of three separate groups of G-quadruplex-interactive compounds and their interaction with G-quadruplex DNA. Using the first group of compounds (anthraquinones), we describe experiments that provide the proof of concept that a G-quadruplex is required for inhibition of telomerase. Using the second group of compounds (perylenes), we describe the structure of a G-quadruplex-ligand complex and its effect on the dynamics of formation and enzymatic unwinding of the quadruplex. For the third group of compounds (porphyrins), we describe the experiments that relate the biological effects to their interactions with G-quadruplexes.
• Rha, S. Y., Izbicka, E., Lawrence, R., Davidson, K., Sun, D., Moyer, M. P., Roodman, G. D., Hurley, L., & Von Hoff, D. (2000). Effect of telomere and telomerase interactive agents on human tumor and normal cell lines. Clinical cancer research : an official journal of the American Association for Cancer Research, 6(3), 987-93.
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  Shortening of telomeres along with an up-regulation of telomerase is implicated in the immortality of tumor cells. Targeting either telomeres or telomerase with specific compounds has been proposed as an anticancer strategy. Because telomerase activity and telomeres are found in normal cells, telomere or telomerase targeting agents could induce side effects in normal tissues. We evaluated the effects of telomere and telomerase interactive agents in human tumor and normal cell lines to try to determine the potential side effects those agents might induce in patients. Toxicity of the G-quadruplex interactive porphyrins (TMPyP4, TMPyP2) and azidothymidine (AZT) were tested using a cell-counting technique against normal human cell lines (CRL-2115 and CRL-2120, fibroblasts; NHEK-Ad, adult keratinocytes; CCL-241, small intestinal cells; NCM 460, colonic mucosal epithelial cells) and human tumor cell lines (MDA-MB 231 and Hs 578T, breast cancer; SK-N-FI, neuroblastoma; HeLa, cervix cancer; MIA PaCa-2, pancreatic cancer; HT-29 and HCT-116, colon cancer; DU 145, prostatic cancer cell line). Telomerase activity of these cell lines was measured by a non-PCR-based conventional assay. The effects of TMPgammaP2, TMPyP4, and AZT were also evaluated against normal human bone marrow specimens, using a granulocyte-macrophage colony-forming assay (CFU-GM). AZT showed very low cytotoxic effects against normal and tumor cell lines, with the IC50 values above 200 microM. The IC50 values for TMPyP2 and TMPyP4 in normal human cell lines were in the range of 2.9-48.3 microM and 1.7-15.5 microM, respectively, whereas in tumor cell lines the IC50 values were 11.4-53 microM and 9.0-28.2 microM, respectively. Within the tissue types, keratinocytes were more sensitive to TMPyP4 than fibroblasts, and small intestinal cells were more sensitive than colonic mucosal epithelial cells. The IC50 for TMPyP2 and TMPyP4 in the normal marrow colony-forming assays were 19.3 +/- 5.1 microM and 47.9 +/-1.0 microM, respectively. In conclusion, the in vitro cytotoxicity of the telomere interactive agent TMPyP4 is comparable in human tumor and normal cell lines, which indicates that TMPyP4 could have effects on normal tissues.
• Cathers, B. E., Sun, D., & Hurley, L. H. (1999). Accurate determination of quadruplex binding affinity and potency of G-quadruplex-interactive telomerase inhibitors by use of a telomerase extension assay requires varying the primer concentration. Anti-cancer drug design, 14(4), 367-72.
• Izbicka, E., Wheelhouse, R. T., Raymond, E., Davidson, K. K., Lawrence, R. A., Sun, D., Windle, B. E., Hurley, L. H., & Von Hoff, D. D. (1999). Effects of cationic porphyrins as G-quadruplex interactive agents in human tumor cells. Cancer research, 59(3), 639-44.
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  A series of cationic porphyrins has been identified as G-quadruplex interactive agents (QIAs) that stabilize telomeric G-quadruplex DNA and thereby inhibit human telomerase; 50% inhibition of telomerase activity was achieved in HeLa cell-free extract at porphyrin concentrations in the range < or = 50 microM. Cytotoxicity of the porphyrins in vitro was assessed in normal human cells (fibroblast and breast) and human tumor cells representing models selected for high telomerase activity and short telomeres (breast carcinoma, prostate, and lymphoma). In general, the cytotoxicity (EC50, effective concentration for 50% inhibition of cell proliferation) against normal and tumor cells was > 50 microM. The porphyrins were readily absorbed into tumor cell nuclei in culture. Inhibition of telomerase activity in MCF7 cells by subcytotoxic concentrations of TMPyP4 showed time and concentration dependence at 1-100 microM TMPyP4 over 15 days in culture (10 population doubling times). The inhibition of telomerase activity was paralleled by a cell growth arrest in G2-M. These results suggest that relevant biological effects of porphyrins can be achieved at concentrations that do not have general cytotoxic effects on cells. Moreover, the data support the concept that a rational, structure-based approach is possible to design novel telomere-interactive agents with application to a selective and specific anticancer therapy.
• Kwok, Y., Sun, D., Clement, J. J., & Hurley, L. H. (1999). The quinobenzoxazines: relationship between DNA binding and biological activity. Anti-cancer drug design, 14(5), 443-50.
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  The quinobenzoxazine compounds, derived from antibacterial quinolones, is active in vitro and in vivo against murine and human tumors. In this contribution, we show that the relative DNA binding affinity of the quinobenzoxazine compounds correlates with their cytotoxicity, their ability to inhibit gyrase-DNA complex formation, and the decatenation of kinetoplast DNA by human topoisomerase II. DNA binding studies with the descarboxy-A-62176 analogue indicate that the beta-keto acid moiety of the quinobenzoxazine compounds plays an important role in their interaction with DNA.
• Raymond, E., Sun, D., Izbicka, E., Mangold, G., Silvas, E., Windle, B., Sharma, S., Soda, H., Laurence, R., Davidson, K., & Hoff, D. V. (1999). A human breast cancer model for the study of telomerase inhibitors based on a new biotinylated-primer extension assay. British Journal of Cancer, 80(9), 1332-1341.
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  PMID: 10424733;PMCID: PMC2363066;Abstract: Telomerase is an RNA-dependent polymerase that synthesizes telomeric DNA (TTAGGG)(n) repeats. The overall goal of our work was to establish human cancer models that can be used to design clinical trials with telomerase inhibitors. The objectives of this study were (1) to set up a human breast cancer system that allows evaluation of the effects of telomerase inhibitors in cultured cells using a non-amplified telomerase assay and (2) to test this system using two drugs (cisplatin and TMPyP4) that affect the telomerase expression in breast cancer cells in culture. We first compared the telomerase activity in a variety of human breast cancer cell lines to that of other tumour types using a new biotinylated-primer extension assay. Our method, based on a non-amplified primer extension assay shows the direct incorporation of 32P-labelled nucleotides induced by telomerase on human telomeric primers. The 32P-dGTP labelled telomerase-extended 5'-biotinylated (TTAGGG)3 primer can subsequently be separated using streptavidin-coated magnetic beads. As compared to other non-amplified method, we showed that this procedure improved the characterization and the quantification of the banding pattern resulting from telomerase extension by reducing the radioactive background. Using this method, we observed that telomerase activity varies markedly in a panel of 39 human cancer cell lines. For example, MCF7 breast cancer cells in culture showed intermediate telomerase activity corresponding to 33.8 ± 3.4% of that of the HeLa cells (reference cell line). Similarly, the telomere length varied with each cell line (average: 6.24 ± 6.16). No correlation between the level of telomerase and telomere length was observed, suggesting that a high processivity is not required to maintain telomeres and that, in some cell lines, another mechanism of telomere elongation can maintain telomere length. From this study, we selected MCF7 and MX1 models that showed reproducible telomerase activity and a relatively limited telomere length for the testing of potential telomere-telomerase interacting agents. Using cisplatin and a new porphyrin-derived compound TMPyP4, we showed that our model was able to detect a down-regulation of the telomerase activity in MCF7 cells in culture and in a human MX1 tumour xenografts. Based on these results, a breast cancer model for evaluating telomerase and telomere interactive agents is proposed.
• Sun, D., Lopez-Guajardo, C. C., Quada, J., Hurley, L. H., & Von Hoff, D. D. (1999). Regulation of catalytic activity and processivity of human telomerase. Biochemistry, 38(13), 4037-44.
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  The ends of eukaryotic chromosomes are specialized sequences, called telomeres comprising tandem repeats of simple DNA sequences. Those sequences are essential for preventing aberrant recombination and protecting genomic DNA against exonucleolytic DNA degradation. Telomeres are maintained at a stable length by telomerase, an RNA-dependent DNA polymerase. Recently, human telomerase has been recognized as a unique diagnostic marker for human tumors and is potentially a highly selective target for antitumor drugs. In this study, we have examined the major factors affecting the catalytic activity and processivity of human telomerase. Specifically, both the catalytic activity and processivity of human telomerase were modulated by temperature, substrate (dNTP and primer) concentration, and the concentration of K+. The catalytic activity of telomerase increased as temperature (up to 37 degrees C), concentrations of dGTP, primer, and K+ were increased. However, the processivity of human telomerase decreased as temperature, primer concentration, and K+ were increased. Our results support the current model for human telomerase reaction and strengthen the hypothesis that a G-quadruplex structure of telomere DNA plays an important role in the regulation of the telomerase reaction.
• Fletcher, T. M., Sun, D., Salazar, M., & Hurley, L. H. (1998). Effect of DNA secondary structure on human telomerase activity. Biochemistry, 37(16), 5536-41.
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  Telomeres are specialized DNA-protein complexes located at the chromosome ends. The guanine-rich telomeric sequences have the ability to form G-quadruplex structures under physiological ionic conditions in vitro. Human telomeres are maintained through addition of TTAGGG repeats by the enzyme telomerase. To determine a correlation between DNA secondary structure and human telomerase, telomerase activity in the presence of various metal cations was monitored. Telomerase synthesized a larger proportion of products corresponding to four, five, eight, and nine full repeats of TTAGGG in 100 mM K+ and to a lesser extent in 100 mM Na+ when a d(TTAGGG)3 input primer was used. A more even product distribution was observed when the reaction mixture contained no added Na+ or K+. Increasing concentrations of Cs+ resulted in a loss of processivity but not in the distinct manner observed in K+. When the input primer contained 7-deaza-dG, the product distribution resembled that of reactions without K+ even in the presence of 100 mM K+. Native polyacrylamide gel electrophoresis indicated that d(TTAGGG)4, d(TTAGGG)5, d(TTAGGG)8, and d(TTAGGG)9 formed compact structures in the presence of K+. The oligonucleotide d(TTAGGG)4 had a UV spectrum characteristic of that of the G-quadruplex only in the presence of K+ and Na+. A reasonable explanation for these results is that four, five, eight, and nine repeats of TTAGGG form DNA secondary structures which promote dissociation of the primer from telomerase. This suggests that telomerase activity in cells can be modulated by the secondary structure of the DNA template. These findings are of probable relevance to the concept of telomerase as a therapeutic target for drug design.
• Sun, D., Hurley, L. H., & Von Hoff, D. D. (1998). Telomerase assay using biotinylated-primer extension and magnetic separation of the products. BioTechniques, 25(6), 1046-51.
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  Human telomerase, a ribonucleoprotein enzyme is known to be associated with immortalized cancer cells but is absent in most normal tissues. Thus, telomerase appears to be an attractive new target for anticancer agents and an important diagnostic marker of human cancers. Here, we describe an improved telomerase assay method based on the Dynabead biomagnetic separation theory. In this method, 5'-biotinylated (TTAGGG)3 was used as a primer for the telomerase reaction. Telomerase reaction products were then immobilized on streptavidin-coated Dynabeads and washed intensively to eliminate excess [alpha-32P]dGTP. Using this method, without the amplification of telomerase reaction products by the PCR, we were able to quantitatively detect telomerase activity in human HeLa cell extracts equivalent to between 200-500 cells. This method is anticipated to be useful for the measurement of telomerase activity in various tumor cells, for assessing potential telomerase and for understanding the biochemical aspects of the telomerase reaction.
• Sharma, S., Raymond, E., Soda, H., Sun, D., Hilsenbeck, S. G., Sharma, A., Izbicka, E., Windle, B., & Hoff, D. V. (1997). Preclinical and clinical strategies for development of telomerase and telomere inhibitors. Annals of Oncology, 8(11), 1063-1074.
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  PMID: 9426325;Abstract: Background: Telomerase is an important enzyme whose activity has been convincingly demonstrated in humans recently. It is required for maintenance of ends of chromosomes (telomeres) during cell division. Since its presence has been selectively demonstrated in dividing cells including tumor cells, it has generated considerable excitement as a potential anti-cancer strategy. Design: In this article, we review the current relevant biology of the enzyme, the challenges encountered in the preclinical phase of target development and the current efforts that focus on telomeres and telomerase as therapeutic targets. We also speculate on the potential toxicities and mechanisms of resistance that may be encountered during use of such therapies.
• Sun, D., Thompson, B., Cathers, B. E., Salazar, M., Kerwin, S. M., Trent, J. O., Jenkins, T. C., Neidle, S., & Hurley, L. H. (1997). Inhibition of human telomerase by a G-quadruplex-interactive compound. Journal of medicinal chemistry, 40(14), 2113-6.
• Raymond, E., Sun, D., Chen, S., Windle, B., & D., D. (1996). Agents that target telomerase and telomeres. Current Opinion in Biotechnology, 7(6), 583-591.
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  PMID: 8939642;Abstract: Telomeres are guanine-rich regions that are located at the ends of chromosomes and are essential for preventing aberrant recombination and protecting against exonucleolytic DNA degradation. Telomeres are maintained by telomerase, an RNA-dependent DNA polymerase. Because telomerase is known to be expressed in tumor cells, which concurrently have short telomeres, and not in most somatic cells, which usually have long telomeres, telomerase and telomere structures have been recently proposed as attractive targets for the discovery of new anticancer agents. The most exciting current strategies are aimed at specifically designing new drugs that target telomerase or telomeres and new models have been formulated to study the biological effects of inhibitors of telomerase and telomeres both in vitro and in vivo.
• Sun, D., Hurley, L. H., & Harshey, R. M. (1996). Structural distortions induced by integration host factor (IHF) at the H' site of phage lambda probed by (+)-CC-1065, pluramycin, and KMnO4 and by DNA cyclization studies. Biochemistry, 35(33), 10815-27.
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  Integration Host Factor (IHF) is a sequence-specific DNA-bending protein that is proposed to interact with DNA primarily through the minor groove. We have used various chemical probes [(+)-CC-1065, a minor-groove-specific agent that alkylates N3 of adenine and traps bends into the minor groove; pluramycin, a minor-major-groove threading intercalator that alkylates N7 of guanine; KMnO4, which reacts more strongly with bases in denatured DNA] to gain more information on the interaction of IHF with the H' site of phage lambda. In addition to the 13-bp core consensus recognition element present at all IHF binding sites, the H' site also has an upstream AT-rich element that increases the affinity of IHF for this site. Our results reveal new details of IHF-DNA interaction at this site. Results with (+)-CC-1065 modification suggest that IHF interacts with the adenines on the 3'-side of the AT-rich element and likely induces a minor-groove bend in its vicinity, which in turn stabilizes the interaction. Pluramycin modification experiments suggest the presence of both short- and long-range structural perturbations (possible DNA unwinding events) on either side of the IHF contact region. Although IHF is known to induce a large bend in DNA at the H' site, no separation of base pairs was detected when the bent DNA was probed with KMnO4. DNA cyclization studies indicate a large magnitude (approximately 180 degrees) for the IHF-induced bend at the H' site, consistent with > 140 degrees bend estimated by gel electrophoresis methods. These studies suggest that IHF-induced DNA bending is accompanied by the introduction of a DNA node, DNA unwinding, and/or by some other DNA distortion. An enhanced binding and stability of IHF was observed on small circular DNA.
• Fan, J. Y., Sun, D., Yu, H., Kerwin, S. M., & Hurley, L. H. (1995). Self-assembly of a quinobenzoxazine-Mg2+ complex on DNA: a new paradigm for the structure of a drug-DNA complex and implications for the structure of the quinolone bacterial gyrase-DNA complex. Journal of medicinal chemistry, 38(3), 408-24.
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  The quinobenzoxazine compounds A-62176 and A-85226 belong to a novel class of antineoplastic agents that are catalytic inhibitors of topoisomerase II and also structural analogs of the antibacterial DNA gyrase inhibitor Norfloxacin. In vitro studies have shown that their antineoplastic activity is dependent upon the presence of divalent metal ions such as Mg2+ and Mn2+, although the precise role of these ions in the mechanism of action is unknown. In this study we have investigated the structures of the binary complex between the quinobenzoxazines and Mg2+ and the ternary complex between quinobenzoxazine-Mg2+ and DNA. The stoichiometry of the binary and ternary complexes and the biophysical studies suggest that a 2:2 drug:Mg2+ complex forms a "heterodimer complex" with respect to DNA in which one drug molecule is intercalated into DNA and the second drug molecule is externally bound, held to the first molecule by two Mg2+ bridges, which themselves are chelated to phosphates on DNA. There is a cooperativity in binding of the quinobenzoxazines to DNA, and a 4:4 drug:Mg2+ complex is proposed in which the two externally bound molecules from two different 2:2 dimers interact via pi-pi interactions. The externally bound quinobenzoxazine molecules can be replaced by the quinolone antibacterial compound Norfloxacin to form mixed-structure dimers on DNA. Based upon the proposed model for the 2:2 quinobenzoxazine:Mg2+ complex on DNA, a parallel model for the antibacterial quinolone-Mg2(+)-DNA gyrase complex is proposed that relies upon the ATP-fueled unwinding of DNA by gyrase downstream of the cleavable complex site. These models, which have analogies to leucine zippers, represent a new paradigm for the structure of drug-DNA complexes. In addition, these models have important implications for the design of new gyrase and topoisomerase II inhibitors, in that optimization for structure-activity relationships should be carried out on two different quinolone molecules rather than a single molecule.
• Sun, D., & Hurley, L. H. (1995). TBP binding to the TATA box induces a specific downstream unwinding site that is targeted by pluramycin. Chemistry & biology, 2(7), 457-69.
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  The TATA-binding protein (TBP) is one of the major components of the human TFIID multiprotein complex. It is important in directing the initiation of RNA transcription at a site immediately downstream of the TATA sequence (TATA box) found in many eukaryotic promoters. The crystal structure of TBP complexed with an oligonucleotide containing the TATA box revealed a protein with an approximate two-fold symmetry which apparently has symmetrical interactions with DNA. It is not known how an asymmetric effect involving downstream activation can be produced by an apparent symmetric complex. We set out to examine the state of DNA in the TBP-DNA complex using pluramycin, a small molecular weight probe of DNA accessibility.
• Sun, D., Hansen, M., & Hurley, L. H. (1995). Molecular Basis for the DNA Sequence Specificity of the Pluramycins. A Novel Mechanism Involving Groove Interactions Transmitted through the Helix via Intercalation To Achieve Sequence Selectivity at the Covalent Bonding Step. Journal of American Chemical Society, 117, 2340-2440. doi:10.1021/ja00114a007
• Thompson, A. S., Fan, J. Y., Sun, D., Hansen, M., & Hurley, L. H. (1995). Determination of the structural role of the internal guanine-cytosine base pair in recognition of a seven-base-pair sequence cross-linked by bizelesin. Biochemistry, 34(35), 11005-16.
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  Bizelesin (formerly U77,779, The Upjohn Co.) is a bifunctional DNA cross-linking antitumor antibiotic consisting of two open-ring homologs of the (+)-CC-1065 cyclopropa[c]pyrrolo[3,2-e]indol-4(5H)-one (CPI) subunits connected by a rigid linking moiety. Previous studies have shown that Bizelesin most often forms an interstrand cross-link through the N3 of two adenines 6 base pairs (bp) apart (inclusive of the modified adenines). However, gel electrophoresis studies have also indicated that Bizelesin forms 7-bp cross-links in specific sequences. In most of these sequences the cross-linked adenines represent the only possible cross-link site (i.e., no 6-bp site is available); however, in several sequences, a 7-bp sequence is selected in overwhelming preference to a possible 6-bp sequence. In this study, we demonstrate the unique requirement for a G.C base pair within this sequence and the critical presence of the exocyclic 2-amino group of guanine. In a subsequent two-dimensional 1H-NMR study that concentrates on the 7-bp cross-link formed with the sequence 5'-TTAGTTA-3', the role of the central G.C base pairs in the formation of a 7-bp cross-link is probed. 1H-NMR analysis coupled with restrained molecular dynamics (rMD) provides evidence for distortion around the covalently modified adenines. Because of this distortion, the modified bases are twisted toward the center of the duplex adduct, effectively reducing the cross-linked distance. The rMD study also indicates that a hydrogen bond is formed between the exocyclic amine of the central guanine and the carbonyl of the ureylene linker. On the basis of the observation of the distortion in the duplex and the hydrogen bonding between the drug and DNA, it is possible to speculate on the role of the central G.C bases in this sequence preference and propose a mechanism by which Bizelesin forms a 7-bp rather than a 6-bp cross-link with this sequence.
• Thompson, A. S., Sun, D., & Hurley, L. H. (1995). Monoalkylation and Crosslinking of DNA by Cyclopropylpyrrolindoles Entraps Bent and Straight Forms of A-Tracts. Journal of American Chemical society, 117, 2371-2372. doi:10.1021/ja00113a035
• Hurley, L. H., & Sun, D. (1994). (+)-CC-1065 as a probe for intrinsic and protein-induced bending of DNA. Journal of molecular recognition : JMR, 7(2), 123-32.
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  (+)-CC-1065 is a biologically potent DNA-reactive antitumor antibiotic produced by Streptomyces zelensis. This antibiotic covalently modifies DNA by alkylation of N-3 of adenine in the minor groove. As a structural consequence of covalent modification of DNA, the helix axis is bent into the minor groove. The drug-induced bending of DNA has similarities to intrinsic A-tract bending and the 3' adenine of A-tracts shows a unique reactivity to alkylation by (+)-CC-1065. Upon covalent modification of A-tracts, the magnitude of bending is increased and the helix is stiffened. Using high-field NMR, hydroxyl-radical footprinting and gel electrophoresis, the molecular basis for the high reactivity of the bonding sequence 5'-AGTTA* (an asterisk indicates the covalent modification site) to (+)-CC-1065 has been shown to involve the inherent conformational flexibility of this sequence. Furthermore, these studies also demonstrate that after alkylation the drug-induced bending is focused over the TT region. By analogy with the junction bend model for A-tracts, a 'truncated junction bend model' is proposed for this structure. Last, the application (+)-CC-1065 entrapped/induced bending of DNA as a probe for the Sp1-induced bending of the 21-base-pair repeat and Mu transposase bending of the att L3 sequence is described.
• Sun, D., & Hurley, L. H. (1994). Binding of Sp1 to the 21-bp repeat region of SV40 DNA: effect of intrinsic and drug-induced DNA bending between GC boxes. Gene, 149(1), 165-72.
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  The effect of the antitumor antibiotic (+)-CC-1065 on the binding of Sp1 to the 21-bp repeats of SV40 DNA has been investigated. (+)-CC-1065 alkylates N3 of adenine in DNA and resides in the minor groove. As a consequence of alkylation of the two 5'-AGTTA* sequences (* indicates covalent modification site), which reside between GC boxes III and IV, and boxes V and VI, protein binding to the 3' sites is completely abolished and there is a significant decrease in Sp1 binding to the other regions. The effect of substituting A5 tracts for the (+)-CC-1065-bonding sequence was intermediate between the unmodified 5'-AGTTA* and the drug-modified sequences. It is proposed that a structural distortion of DNA associated with stiffening of the helix induced by the drug-adduct formation is primarily responsible for the inhibition of binding of Sp1 molecules to 21-bp repeats, rather than steric hindrance due to the occupancy by drug molecules of the minor groove within that region.
• Sun, D., & Hurley, L. H. (1994). Cooperative bending of the 21-base-pair repeats of the SV40 viral early promoter by human Sp1. Biochemistry, 33(32), 9578-87.
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  The overall structural features of the multimeric complex between Sp1 and the 21-base-pair repeat of the early promoter region of SV40 DNA have been determined using hydroxyl-radical footprinting; (+)-CC-1065, a sequence-specific minor groove bending probe; and circularization experiments. The results show that the 21-base-pair repeat region has an intrinsically in-phase bent structure that is stabilized upon saturation Sp1 binding by protein-DNA and protein-protein interactions to produce a looping structure. The direction of the Sp1-stabilized bending of DNA occurs into the minor groove and is localized between each of the Sp1 binding sites. These results are used as the basis to propose a looping structure for the multimeric Sp1 21-base-pair repeat region of SV40 DNA. Last, these results provide a rationale for the recently observed inhibition of basal transcriptional levels by site-specific triple-helical DNA complexes.
• Aristoff, P. A., Johnson, P. D., Sun, D., & Hurley, L. H. (1993). Synthesis and biochemical evaluation of the CBI-PDE-I-dimer, a benzannelated analog of (+)-CC-1065 that also produces delayed toxicity in mice. Journal of medicinal chemistry, 36(14), 1956-63.
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  A practical synthesis of CBI (2) was developed and applied to the synthesis of benzannelated analogs of CC-1065, including CBI-PDE-I-dimer (13) and CBI-bis-indole [(+)-A'BC]. The CBI-PDE-I-dimer was shown to have similar DNA sequence selectivity and structural effects on DNA as (+)-CC-1065. Of particular importance was the observed duplex winding effect that has been associated with the pyrrolidine ring of the nonalkylated subunits of (+)-CC-1065 and possibly correlated with its delayed toxicity effects. The effect of CBI-PDE-I-dimer was also compared to (+)-CC-1065 in the inhibition of duplex unwinding by helicase II and nick sealing by T4 ligase and found to be quantitatively similar. The in vitro and in vivo potencies of the CBI compounds corresponded very closely to the corresponding CPI derivatives. Finally, CBI-PDE-I-dimer was like (+)-CC-1065 in causing delayed toxicity in mice.
• Sun, D., & Hurley, L. H. (1993). Analysis of the monoalkylation and cross-linking sequence specificity of bizelesin, a bifunctional alkylation agent related to (+)-CC-1065. Journal of American Chemical Society, 115, 5925-5933. doi:10.1021/ja00067a005
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  The sequence specificity of bizelesin, an interstrand DNA-DNA cross-linker related to the monoalkylating compound (+)-CC-1065, was studied using restriction enzyme fragments. Bizelesin, like (+)-CC-1065, forms monoalkylation adducts through N3 of adenine but can also form DNA-DNA cross-links six base pairs apart on opposite strands. Compared to many other minor groove cross-linking compounds, bizelesin is very efficient at cross-linking DNA. There is a higher than expected proportion of cross-linked adducts based upon the relative number of cross-linked vs monoalkylated adducts. This is rationalized based upon the relative thermodynamic stability of the cross-linked vs monoalkylated species. Where bizelesin monoalkylation occurs, the sequence specificity is significantly higher than those of (+)-CC-1065 and other monoalkylating (+)-CPI analogs. The bizelesin GC tolerance at cross-linking sites is twice as high as for the monoalkylation sites. This increased GC tolerance can be largely explained by the covalent immobilization of the second alkylation arm at sequences that are not normally reactive toward CPI monoalkylation compounds but are made reactive due to a proximity effect. This same rationale can be used to explain the reactivity of the second alkylation arm of bizelesin with guanine, cytosine, and thymine on some sequence. There are some sequences that appear to be unusual in their reactivity with bizelesin in that bizelesin formed cross-linking spanning seven base pairs, and bizelesin forms monoakylation adducts on guanine. In these cases, it is proposed that bizelesin may trap out rare conformational forms during the second alkylation step, or bizelesin may alkylate unusual sites due to the strong precovalent affinity of bizelesin for those sites. Analysis of the monoalkylation and cross-linking sequence specificity of bizelesin, a bifunctional alkylation agent related to (+)-CC-1065. Available from: https://www.researchgate.net/publication/231435650_Analysis_of_the_monoalkylation_and_cross-linking_sequence_specificity_of_bizelesin_a_bifunctional_alkylation_agent_related_to_-CC-1065 [accessed Feb 7, 2016].
• Sun, D., Hansen, M., Clement, J. J., & Hurley, L. H. (1993). Structure of the altromycin B (N7-guanine)-DNA adduct. A proposed prototypic DNA adduct structure for the pluramycin antitumor antibiotics. Biochemistry, 32(32), 8068-74.
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  Altromycin B belongs to the pluramycin family of antitumor antibiotics, which also includes kidamycin, hedamycin, pluramycin, neopluramycin, DC92-B, and rubiflavin A. These potent antitumor compounds react with DNA in as yet imprecisely determined ways. In the present investigation, we have used gel electrophoresis methods in combination with nuclear magnetic resonance and mass spectrometry to determine the structure of the altromycin B-DNA adduct. High-resolution gel electrophoresis demonstrated that guanine was the reactive base, and N7 was implicated from experiments in which N7-deazaguanine was used in place of guanine in a strand breakage assay. Experiments using supercoiled DNA demonstrated that altromycin B and related drugs intercalated into DNA, which implicated this as a common mechanism for binding of the pluramycin antibiotics to DNA. The altromycin B-guanine adduct was isolated from calf thymus DNA after thermal depurination of the alkylated DNA. Mass spectrometry confirmed that altromycin alkylated DNA through guanine, and 1H- and 13C-NMR was used to confirm the covalent linkage sites between altromycin B and guanine. On the basis of these results, we propose that altromycin B first intercalates into DNA via a threading mechanism, reminiscent of nogalamycin, to insert the disaccharide into the minor groove and position the epoxide in the major groove in proximity to N7 of guanine. Nucleophilic attack from N7 of guanine leads to an acid-catalyzed opening of the epoxide, resulting in the altromycin B-DNA adduct. On the basis of these results, a general mechanism for the interaction of the pluramycin family of antibiotics with DNA is proposed.
• Sun, D., Lin, C. H., & Hurley, L. H. (1993). A-tract and (+)-CC-1065-induced bending of DNA. Comparison of structural features using non-denaturing gel analysis, hydroxyl-radical footprinting, and high-field NMR. Biochemistry, 32(17), 4487-95.
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  (+)-CC-1065 is a biologically potent DNA-reactive antitumor antibiotic produced by Streptomyces zelensis. In a previous study we have reported that (+)-CC-1065 produces bending of DNA that has similarities to that intrinsically associated with A-tracts [Lin, C. H., Sun, D., & Hurley, L. H. (1991) Chem. Res. Toxicol, 4, 21-26]. In this article we provide evidence using a combination of non-denaturing gel analysis, hydroxyl-radical footprinting, and high-field NMR for both distinctions between the two types of bends and the importance of junctions in both types of bends. For A-tracts we demonstrate that the locus of bending is at the center of an A-tract and that upon modification of the 3' adenine with (+)-CC-1065 this locus is moved less than 1 base pair to the 3' side, and the bending magnitude is significantly increased. For drug bonding sequences such as 5'-AGTTA* or 5'-GATTA* (where * denotes the drug bonding site), the locus of bending is found to be between the two thymines, and the bending is focused over a 2-base-pair sequence rather than a 5-base-pair sequence, as is the case for the A-tract. An important distinction between an A-tract intrinsic bend and a (+)-CC-1065-induced bend is the effect of temperature. While, as shown previously, the magnitude of A-tract bending increases with decrease in temperature, for drug-induced bending of 5'-AGTTA* the bending magnitude increases with increased temperature.(ABSTRACT TRUNCATED AT 250 WORDS)
• Sun, D., Park, H. J., & Hurley, L. H. (1993). Alkylation of guanine and cytosine in DNA by bizelesin. Evidence for a covalent immobilization leading to a proximity-driven alkylation of normally unreactive bases by a (+)-CC-1065 cross-linking compound. Chemical research in toxicology, 6(6), 889-94.
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  Bizelesin, an intrahelical DNA-DNA interstrand cross-linker related to (+)-CC-1065, has been shown to alkylate DNA through guanine in restriction enzyme sequences in which there is a suitably positioned adenine contained in a highly reactive monoalkylation sequence on the opposite strand. Oligomers containing the sequence 5'-TTTTTN*, in which "N" was either G, C, or T, were synthesized to evaluate the cross-linking potential of bizelesin at nonadenine bases. Kinetic analysis of monoalkylation and cross-linking events demonstrates that it is the reaction at "N" (guanine or cytosine) that results in the cross-link which is the slow step. On the basis of this analysis and the normal unreactivity of guanine and cytosine to alkylation by the cyclopropapyrroloindole alkylating moiety of (+)-CC-1065, we propose that the molecular mechanism for this type of cross-linking reaction most likely involves a covalent immobilization of the second alkylating arm, resulting in a "proximity-driven" reaction.
• Maine, I. P., Sun, D., Hurley, L. H., & Kodadek, T. (1992). The antitumor agent CC-1065 inhibits helicase-catalyzed unwinding of duplex DNA. Biochemistry, 31(16), 3968-75.
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  The antitumor drug CC-1065 is thought to exert its effects by covalent bonding to N3 of adenine in DNA and interfering with some aspect of DNA metabolism. Therefore, it is of interest to determine what effect this drug has on enzymes involved in various aspects of DNA metabolism. In this report, we examine the ability of two DNA helicases, the dda protein of phage T4 and helicase II of Escherichia coli, to unwind CC-1065-adducted, tailed, oligonucleotides. It is shown that the presence of the drug on DNA strongly inhibits unwinding catalyzed by the T4 and E. coli proteins. A significant difference between the results obtained with the two helicases is that DNAs containing drug on either the tailed or the completely duplex strands are poor substrates for helicase II but dda protein-mediated unwinding is inhibited only when the drug is on the tailed strand. The drug-modified, helicase-released, strands migrate abnormally through a native gel, suggesting that the drug traps an unusual secondary structure generated in the course of protein-mediated unwinding. A kinetic analysis of the drug-inhibited reactions reveals that the helicases are trapped by the DNA-drug complex. This is evidenced by a decrease in the rate of helicase exchange between drug-bound substrate and drug-free duplex. The implications of these results with respect to the mechanism of action of CC-1065 in vivo are discussed.
• Sun, D., & Hurley, L. H. (1992). Effect of the (+)-CC-1065-(N3-adenine)DNA adduct on in vitro DNA synthesis mediated by Escherichia coli DNA polymerase. Biochemistry, 31(10), 2822-9.
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  (+)-CC-1065 is a potent antitumor antibiotic produced by Streptomyces zelensis. Previous studies have shown that the potent cytotoxic and antitumor activities of (+)-CC-1065 are due to the ability of this compound to covalently modify DNA. (+)-CC-1065 reacts with duplex DNA to form an N3-adenine DNA adduct which lies in the minor groove of the DNA helix overlapping with a 5-base-pair region. As a consequence of covalent modification with (+)-CC-1065, the DNA helix bends into the minor groove and also undergoes winding and stiffening [Lee, C.-S., Sun, D., Kizu, R., & Hurley, L. H. (1991) Chem. Res. Toxicol. 4, 203-213]. In the studies described here, in which we have constructed site-directed DNA adducts on single-stranded DNA templates, we have shown that (+)-CC-1065 and select synthetic analogues, which have different levels of cytotoxicity, all show strong blocks against progression of Klenow fragment, E. coli DNA polymerase, and T4 DNA polymerase. The inhibition of bypass of drug lesions by polymerase could be partially alleviated by increasing the concentration of dNTPs and, to a small extent, by increasing polymerase levels. Klenow fragment binds equally well to a DNA template adjacent to a drug modification site and to unmodified DNA. These results taken together lead us to suspect that it is primarily inhibition of base pairing around the drug modification site and not prevention of polymerase binding that leads to blockage of DNA synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)
• Sun, D., & Hurley, L. H. (1992). Inhibition of T4 DNA ligase activity by (+)-CC-1065: demonstration of the importance of the stiffening and winding effects of (+)-CC-1065 on DNA. Anti-cancer drug design, 7(1), 15-36.
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  Non-denaturing gel electrophoresis analysis demonstrates that the stiffening and winding effects of (+)-CC-1065 produce unusual proximal and distal inhibition of T4 DNA ligase-catalysed ligation of covalently modified DNA. (+)-CC-1065 is a potent antitumor antibiotic produced by Streptomyces zelensis. This drug selectively bonds through N3 of adenine in DNA and lies in the minor groove of DNA, reacting in a highly sequence-selective manner. Previous studies (Lee et al., 1991) have shown that (+)-CC-1065 produces bending and winding of DNA. The DNA bending and sequence specificity is mediated by the alkylating 'A' subunit of (+)-CC-1065, while the close van der Waals contacts between the non-alkylating B and C subunits of (+)-CC-1065 and the floor of the minor groove of DNA are responsible for the winding of DNA. Covalent modification of oligomers with (+)-CC-1065 and structurally related drugs leads to preferential inhibition of T4 DNA ligase on the non-covalently modified strand to the 5' side of the covalent adduct site, but enhanced ligation of the covalently modified strand. We speculate that the differential effect on proximal strand ligation is due to a drug-induced winding and helix-stabilizing effect which occurs predominantly to the 5' side of the adduct. In addition to the proximal inhibition of ligation, we also describe a distal inhibition of T4 DNA ligase activity which occurs exclusively with drug-modified oligomers and that, if successful, would result in 180 degrees out-of-phase bent DNA following ligation. In this case, two 25 mers or a 21 plus a 29 mer are inhibited from ligation when modified with (+)-CC-1065. This distal ligation is unique to (+)-CC-1065 and its analogs that cause stiffening of the DNA helix. The (+)-CC-1065-induced stiffening effect was demonstrated using a circularization assay and was found to be associated with the close van der Waals contacts between the inside edge of (+)-CC-1065 and the floor of the minor groove, and also to the benzofuran moiety of (+)-ABC" (Adozelesin), a (+)-CC-1065 analog. We conclude from these studies that the DNA-winding and helix-stabilizing effects of these drug molecules can dramatically affect the efficiency of proximal ligation mediated by T4 DNA ligase, and the unusual helix-stiffening effect of (+)-CC-1065, (+)-AB'C' and (+)-ABC" can stabilize the structure of bent DNA formed by drug modification, which results in distal ligase inhibition.
• Sun, D., & Hurley, L. H. (1992). Structure-activity relationships of (+)-CC-1065 analogues in the inhibition of helicase-catalyzed unwinding of duplex DNA. Journal of medicinal chemistry, 35(10), 1773-82.
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  (+)-CC-1065 is a potent antitumor antibiotic produced by Streptomyces zelensis. Previous studies have shown that the potent cytotoxic and antitumor activities of (+)-CC-1065 are due to the ability of this compound to covalently modify DNA. (+)-CC-1065 reacts with duplex DNA to form a (N3-adenine)-DNA adduct which lies in the minor groove of DNA overlapping with a five base-pair region. As a consequence of covalent modification with (+)-CC-1065, the helix bends into the minor groove and also undergoes winding and stiffening. In the studies described here, we have constructed templates for helicase-catalyzed unwinding of DNA that contain site-directed (+)-CC-1065 and analogue DNA adducts. Using these templates we have shown that (+)-CC-1065 and select synthetic analogues, which have different levels of cytotoxicity, all produce a significant inhibition of unwinding of a 3'-tailed oligomer duplex by helicase II when the displaced strand is covalently modified. However, the extent of helicase II inhibition is much more significant for (+)-CC-1065 and an analogue which also produced DNA winding when the winding effects are transmitted in the opposite direction to the helicase unwinding activity. This observed pattern of inhibition of helicase-catalyzed unwinding of drug-modified templates was the same for a 3'-T-tail, for different duplex region sequences, and with the Escherichia coli rep protein. Unexpectedly, the gel mobility of the displaced drug-modified single strand was dependent on the species of drug attached to the DNA. Last, strand displacement by helicase II coupled to primer extension by E. coli DNA polymerase I showed the same pattern of inhibition when the lagging strand was covalently modified. In addition, the presence of helicase II on single-stranded regions of templates caused the premature termination of primer extension by DNA polymerase. These results are discussed from the perspective that (+)-CC-1065 and its analogues have different effects on DNA structure, and these resulting structural changes in DNA molecules are related to the different in vivo biological consequences caused by these drug molecules.
• Lee, C. S., Sun, D., Kizu, R., & Hurley, L. H. (1991). Determination of the structural features of (+)-CC-1065 that are responsible for bending and winding of DNA. Chemical research in toxicology, 4(2), 203-13.
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  Analysis of the anomalous migration in electrophoretic mobilities of (+)-CC-1065-modified oligomers following ligation reveals that (+)-CC-1065 induces DNA bending and winding of the helix. (+)-CC-1065 is a potent antitumor antibiotic produced by Streptomyces zelensis. This drug selectively bonds covalently to N3 of adenine and lies in the minor groove of DNA, reacting in a highly sequence-selective manner. Structurally, (+)-CC-1065 consists of three subunits: two identical pyrroloindole units (subunits B and C) and a third subunit containing the DNA-reactive cyclopropane ring (subunit A). While the bonding reaction is the main determinant of DNA sequence selectivity of (+)-CC-1065, binding interactions between the inside edge substituents of the B and C subunits and the floor of the minor groove of DNA can modulate or fine tune this sequence selectivity, [Hurley, L. H., Lee, C.-S., McGovern, J. P., Mitchell, M. A., Warpehoski, M. A., Kelly, R. C., & Aristoff, P. A. (1988) Biochemistry 27, 3886-3892]. The A subunit of (+)-CC-1065 is responsible for the bending of DNA, and close van der Waals contacts between the inside edge of (+)-CC-1065 and the floor of the minor groove of DNA cause winding equivalent to about 1 base pair per alkylation site and stiffening of DNA. The magnitude of DNA bending induced by (+)-CC-1065 and related compounds is about 14-19 degrees, which is equivalent to that produced by an adenine-thymine tract of about 5-6 base pairs in length. Experiments using oligomers containing both an adenine tract and a unique (+)-CC-1065 bonding site approximately one helix turn apart demonstrate that the directionality of drug-induced bending is in toward the minor groove and the locus of bending is about 2-3 base pairs to the 5'-side of the covalently modified adenine. A circularization efficiency assay shows that the optimum size of circles produced by (+)-CC-1065 and related drugs is between 168 and 180 base pairs. These results are discussed in relation to the molecular basis of the DNA sequence selectivity of (+)-CC-1065, and the (+)-CC-1065-induced DNA bending is compared with the intrinsic bending associated with adenine tracts. Since (+)-CC-1065 induces effects on local DNA structure that appear similar to those produced naturally by adenine tracts and certain DNA binding proteins, the relevance of this phenomenon to biological effects of (+)-CC-1065 and related drugs is considered.
• Lin, C. H., Sun, D. Y., & Hurley, L. H. (1991). (+)-CC-1065 produces bending of DNA that appears to resemble adenine/thymine tracts. Chemical research in toxicology, 4(1), 21-6.
• Kim, S., Rhee, Y., Sun, D., & Yoo, I. (1988). Mutagenesis of Slow Growing Rhizobium japonicum by Transposon Tn5. The Korean Journal of Microbiology, 26(4), 305-311.
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  The spectinomycin resistant strain of slow growing R. japonicum R-168 was selected to be participated in conjugation with E. coli WA803/pGS9. Tn5 was introduced from suicide vector pGS9 into R. japonicum R-168 chromosome at the frequency of and the transconjugante were selected on the yeast extract-mannitol plate containing kanamycin (g/ml) and spectinomycin (g/ml) after 8-9 days incubation. All transconjugants we tested were found to contain Tn 5 DNA on their genome, which was confirmed by Southern hybridization experiments. R. japonicum RNa75, which had been selected through plant test, was found to be defective in symbiotic nitrogen fixing ability and the production of leghemoglobin in soybean nodules formed by the inoculation of this mutant. In addition, this mutant strain hardly developed nitrogenase activity asymbiotically in contrast with the wild type strain, indicating that some nitrogen fixing gene might be blocked in this strain and the production of leghemoglobin could be decreased by the interference in nitrogen fixing genes.
• Sun, D., & Yoo, O. J. (1988). Purification and Characterization of a Restriction Endonuclease Zan I from Zymomonas anaerobia. Korean Biochemical Journal, 21(4), 419-422.
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  We described the purification and characterization of a sequence specific restriction edonuclease, Zan I, found from Zymomonas Anaerobia (NCI B8227). The purified enzyme is essentially homogeneous and the subunit molecular weight is 30,000 +- 1,000 as judged by 10% polyacrylamide gel electrophoresis containing 0.1% SDS. The recognition sequence and the cleavage site (indicated by the arrow) were determined to be 5'-CCl(AT) GG-3', the same sequence of BstN I and EcoR II. Zan I endonuclease is able to cleave dcm-mthylated DNA and is maximally active at the temperature of 37 °C .

Reviews

• Sun, D. (2013. Current and Future Views in G-Quadruplex Secondary Structure as an Anti-Cancer Target(pp e117).