Hurley, L., Sun, D., Guo, K., Rusche, J. J., & Hurley, L. -. (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).
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., Hansen, M., & Hurley, L. (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 the American Chemical Society, 117(9), 2430-2440.
Abstract:
The pluramycin antitumor antibiotics, which include the altromycins, pluramycin, hedamycin, and rubiflavin, are a group of highly evolved DNA-reactive compounds that have structural features reminiscent of both nogalamycin and the aflatoxins. As such, they are characterized as "threading intercalators" with the added ability to alkylate N7 of guanine (see preceding article in this issue). In this article we have demonstrated that different members of this group of antibiotics have sequence specificities that differ for the base pair to the 5′ side of the alkylated guanine and also have a range of reactivities with susceptible sequences. Subsequent experiments were designed to determine the molecular origin for both these observed contrasting sequence specificities and covalent reactivities. First, neopluramycin, an analog of pluramycin that lacks the epoxide, and thus is unable to covalently modify DNA, but is in other respects structurally similar, exhibits no discernible sequence selectivity. This suggests that the sequence selectivity of the pluramycins is determined at the covalent bonding step rather than the precovalent binding interactions. Second, using An tracts of varying length (n = 1-5) to modulate the minor groove geometry to the 5′ side of the covalent alkylation site, this structural parameter has been shown to have a major effect on both sequence specificity and alkylation reactivity. Last, the electronegativity of the N7 position of the alkylated base can also affect reactivity and, to a lesser extent, sequence specificity. In order to determine the molecular details of the interactions in the minor and major grooves, which could give rise to the different sequence specificities of the nonclassical (typified by altromycin B) and classical (typified by hedamycin) pluramycins, we have used molecular models of the altromycin B and hedamycin-DNA adducts that are derived from high-field NMR data of their 10-mer duplex diadducts. These studies demonstrate that it is likely that the sequence-dependent reactivities of the epoxide of the pluramycin to N7 of guanine are dependent upon the relative extent of a "proximity effect". The magnitude of the proximity effect is determined by a "steering reaction", which takes place in the minor and major grooves due to the different placement of the carbohydrate substituents on the pluramycins and their hydrogen bonding and van der Waals interactions with the base pairs to the 5′ side of the alkylation site. This is proposed to be a novel mechanism for sequence recognition, where cooperative interactions in the minor and major grooves transmitted via the intercalation moiety dictate the positioning of the epoxide in the major groove and, thus, sequence reactivity. Finally, we propose that the increased reactivity of the classical pluramycins in contrast to the altromycins is at least partially determined by the "reach" of the reactive epoxide in the major groove, which varies from one group to another. The molecular mechanisms for sequence recognition described here provide a new paradigm for sequence recognition by minor and major groove interactions mediated by intercalactive binding but achieved at the covalent bonding step. * Address correspondence to this author.
Hurley, L. H., & Needham-VanDevanter, D. (1986). Sequence specificity and biological consequences of drugs that bind covalently in the minor groove of DNA.. Basic life sciences, 38, 203-210.
Qin, Y., Fortin, J. S., Tye, D., Gleason-Guzman, M., Brooks, T. A., & Hurley, L. H. (2010). Molecular cloning of the human platelet-derived growth factor receptor β (PDGFR-β) promoter and drug targeting of the g-quadruplex-forming region to repress PDGFR-β expression. Biochemistry, 49(19), 4208-4219.
PMID: 20377208;PMCID: PMC2868086;Abstract:
To understand the mechanisms controlling platelet-derived growth factor receptor β (PDGFR-β) expression in malignancies, we have cloned and characterized the first functional promoter of the human PDGFR-β gene, which has been confirmed by luciferase reporter gene assays. The transcription initiation sites were mapped by primer extension. Promoter deletion experiments demonstrate that the proximal, highly GC-rich region (positions -165 to -139) of the human PDGFR-β promoter is crucial for basal promoter activity. This region is sensitive to S1 nuclease and likely to assume a non-B-form DNA secondary structure within the supercoiled plasmid. The G-rich strand in this region contains a series of runs of three or more guanines that can form multiple different G-quadruplex structures, which have been subsequently assessed by circular dichroism. A Taq polymerase stop assay has shown that three different G-quadruplex-interactive drugs can each selectively stabilize different G-quadruplex structures of the human PDGFR-β promoter. However, in transfection experiments, only telomestatin significantly reduced the human PDGFR-β basal promoter activity relative to the control. Furthermore, the PDGFR-β mRNA level in Daoy cells was significantly decreased after treatment with 1 μM telomestatin for 24 h. Therefore, we propose that ligand-mediated stabilization of specific G-quadruplex structures in the human PDGFR-β promoter can modulate its transcription. © 2010 American Chemical Society.
Kwok, Y., Zeng, Q., & Hurley, L. H. (1998). Topoisomerase II-mediated site-directed alkylation of DNA by psorospermin and its use in mapping other topoisomerase II poison binding sites. Proceedings of the National Academy of Sciences of the United States of America, 95(23), 13531-13536.
PMID: 9811834;PMCID: PMC24853;Abstract:
Psorospermin is a plant natural product that shows significant in vivo activity against P388 mouse leukemia. The molecular basis for this selectivity is unknown, although psorospermin has been demonstrated to intercalate into DNA and alkylate N7 of guanine. Significantly, the alkylation reactivity of psorospermin at specific sites on DNA increased 25- fold in the presence of topoisomerase II. In addition, psorospermin trapped the topoisomerase II-cleaved complex formation at the same site. These results imply that the efficacy of psorospermin is related to its interaction with the topoisomerase H-DNA complex. Because thermal treatment of (N7 guanine)-DNA adducts leads to DNA strand breakage, we were able to determine the site of alkylation of psorospermin within the topoisomerase II gate site and infer that intercalation takes place at the gate site between base pairs at the +1 and +2 positions. These results provide not only additional mechanistic information on the mode of action of the anticancer agent psorospermin but also structural insights into the design of an additional class of topoisomerase II poisons. Because the alkylation site for psorospermin in the presence of topoisomerase II can be assigned unambiguously and the intercalation site inferred, this drug is a useful probe for other topoisomerase poisons where the sites for interaction are less well defined.
