Conformational Preferences of DNA Strands from the Promoter Region of the c-MYC Oncogene

Abstract

We characterized the conformational preferences of DNA in an equimolar mixture of complementary G-rich and C-rich strands from the promoter region of the c-MYC oncogene. Our CD-based approach presupposes that the CD spectrum of such a mixture is the spectral sum of the constituent duplex, G-quadruplex, i-motif, and coiled conformations. Spectra were acquired over a range of temperatures at different pHs and concentrations of KCl. Each spectrum was unmixed in terms of the predetermined spectra of the constituent conformational states to obtain the corresponding weighting factors for their fractional contributions to the total population of DNA. The temperature dependences of those contributions then were analyzed in concert according to a model based on a thermodynamic representation of the underlying equilibria. Fitted estimates of the melting enthalpy and temperature obtained for the duplex, G-quadruplex, and i-motif imply that the driving force behind dissociation of the duplex and the concomitant formation of tetrahelical structures is the folding of the G-strand into the G-quadruplex. The liberated C-strand adopts the i-motif conformation at acidic pH and exists in the coiled state at neutral pH. The i-motif alone cannot induce dissociation of the duplex even at pH 5.0, at which it is most stable. Under the physiological conditions of neutral pH, elevated potassium, and room temperature, the duplex and G-quadruplex conformations coexist with the C-strand in the coiled state. Taken together, our results suggest a novel, thermodynamically controlled mechanism for the regulation of gene expression.

Volumetric Interplay between the Conformational States Adopted by Guanine-Rich DNA from the c-MYC Promoter

“…Figure shows the CD spectra of the G-quadruplex (G-strand at 1 mM KCl), duplex (an equimolar mixture of G- and C-strands at 50 mM CsCl in the absence of KCl), and coiled states of G- and C-strands (50 mM CsCl in the absence of KCl) at 25 °C. The spectra presented in Figure are similar to those we have reported previously for the same states . The observed similarity suggests that DNA strands adopt the respective states at the chosen experimental conditions.…”

“…The observed similarity suggests that DNA strands adopt the respective states at the chosen experimental conditions. In particular, the equimolar ratio of G- and C-strands forms a duplex, while G-strand forms a parallel G-quadruplex in the presence of K + . ,, …”

“…In particular, the equimolar ratio of G-and C-strands forms a duplex, while G-strand forms a parallel G-quadruplex in the presence of K + . 37,55,56 In Figure 2, we plotted the pressure dependences of Gquadruplex-to-coil (at 100 μM KCl) and duplex-to-single strand (at 50 mM CsCl in the absence of KCl) transition temperatures, T M . We have previously shown that G-strand forms a stable G-quadruplex when the K + -to-DNA ratio becomes equal to the stoichiometric value of 2:1.…”

Heat Capacity Changes (ΔCp) for Interconversions between Differentially-Ordered DNA States within Physiological Temperature Domains: Implications for Biological Regulatory Switches

“…In contrast, relatively few studies have focused on the heat capacity differences between order–order transformations, such as transitions between duplex DNA states and a host of competing non-B DNA secondary structures. This deficiency is of concern because such order–order transitions are proposed to play pivotal roles in various biological processes, such as gene regulation and the development of genetic diseases (e.g., refs − ). Some examples of order–order transformations as possible biological regulatory switches include: The order-to-order transformation studied here, which is implicated in triggering DNA triplet expansion, a threshold event that can result in diseases of the central nervous system. , Palindromic DNA sequences that can undergo order–disorder transitions between fully paired duplex domains and extruded cruciform-like structures; a transformation implicated in recombination mechanisms. , Slippage structures that reflect order–order transformations between fully paired duplexes and out of register, misaligned structures; a feature that has been cited as inducing frameshift mutagenesis. , Order-order transitions between duplex and quadruplex domains at the termini of telomeres; a transformation implicated in being protective of chromosomal stability. , Super helical stress- and/or cation-induced transformations from right- to left-handed helical domains (the so-called B to Z transition), implicated in mechanisms of gene regulation. , As emphasized above, heat capacity data as a function of multiple variables are needed to fully evaluate the driving forces and potential biological impacts of such order-to-order conformational switches between ensembles of different nucleic acid “native”/ordered states.…”

Remodeling of the Conformational Dynamics of Noncanonical DNA Structures by Monomeric and Aggregated α-Synuclein

“…Analogous measurements were carried out with a G-quadruplex construct of the c-Myc oncogene promoter region (c-MycG4). 21 The effects of monomeric and aggregated α-syn on the parallel c-MycG4 structure were similar, but less prominent, probably owing to its higher stability (Figure S9; for details please refer to the paragraph in the SI). From dynamic light scattering experiments, the size of the oligomers of α-syn added has been determined to vary between about 20 and 400 nm with a maximum at about 130 nm (Figure S3).…”