G-quadruplexes: Kinetic stability and effects on the c-KIT promoter

Abstract

In addition to the famous Watson & Crick model for B-form duplex DNA, guanine-rich DNA sequences can self-assemble under certain conditions to form a four-stranded structure known as a G-quadruplex. G-quadruplexes are composed of stacks of Gquartets, in which four guanines are arranged in a square planar array, interacting via eight hydrogen bonds. Monovalent cations especially K+ and Na+ but not Li+ stabilize this structure by binding with the central carbonyl O6 atoms. Bioinformatic databases have revealed potential quadruplex-forming sequences throughout the genome and tandem repeats of guanines are found to accumulate upstream of the transcription initiation site of several proto-oncogenes. The promoter region of the c-kit proto-oncogene contains two potential quadruplex forming sequences. The first part of this work focuses on understanding how the c-kit promoter is regulated by potential G-quadruplex forming structures. We have incorporated 165 base pairs of the c-kit promoter region into a luciferase reporter vector and have constructed several mutant variants of this sequence. Determining the level of luciferase expression of these constructed vectors in HeLa and HCT 116 cells have allowed us to elucidate the effect of quadruplex formation on gene expression. Our results reveal that a decrease in gene expression level is observed from the constructed vectors that carry a very stable quadruplex-forming sequence. In genomic DNA, these putative quadruplex-forming G-rich sequences are normally base paired with their complementary C-rich strands to generate duplex DNA. Structural transitions of B-form DNA (duplex) to non-B-form DNA (quadruplex) require local melting, which is facilitated by negative superhelical tension. We have examined in vitro the effect of DNA supercoiling on the reaction of the c-kit promoter (and some variants of the natural sequences) with three chemical probes KMnO4, DEPC, and DMS. The results demonstrated that negative superhelicity did not significantly affect the formation of G-quadruplex. For the first time, we have used two-dimensional agarose gel electrophoresis to probe topology-dependent structural transitions in the c-kit promoter and some of its modified versions. Our results showed that the constructed vectors that carried the very stable quadruplex-forming sequence undergo unusual structural transition. Finally, we have used a gel based assay to understand the dynamic equilibrium between quadruplex and duplex DNA under defined conditions. The results show that at elevated temperatures, the formation of duplex DNA with these G-rich sequences is kinetically reversible and we have measured the rate at which the duplex strand exchanges with single-stranded DNA. The formation of both quadruplex and duplex DNA are cation and concentration-dependent.

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ORCID for Keith R. Fox: orcid.org/0000-0002-2925-7315

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