Tertiary DNA Structure in the Single-Stranded hTERT Promoter Fragment Unfolds and Refolds by Parallel Pathways via Cooperative or Sequential Events

Abstract

The discovery of G-quadruplexes and other DNA secondary elements has increased the structural diversity of DNA well beyond the ubiquitous double helix. However, it remains to be determined whether tertiary interactions can take place in a DNA complex that contains more than one secondary structure. Using a new data analysis strategy that exploits the hysteresis region between the mechanical unfolding and refolding traces obtained by a laser-tweezers instrument, we now provide the first convincing kinetic and thermodynamic evidence that a higher order interaction takes place between a hairpin and a G-quadruplex in a single-stranded DNA fragment that is found in the promoter region of human telomerase. During the hierarchical unfolding or refolding of the DNA complex, a 15-nucleotide hairpin serves as a common species among three intermediates. Moreover, either a mutant that prevents this hairpin formation or the addition of a DNA fragment complementary to the hairpin destroys the cooperative kinetic events by removing the tertiary interaction mediated by the hairpin. The coexistence of the sequential and the cooperative refolding events provides direct evidence for a unifying kinetic partition mechanism previously observed only in large proteins and complex RNA structures. Not only does this result rationalize the current controversial observations for the long-range interaction in complex single-stranded DNA structures, but this unexpected complexity in a promoter element provides additional justification for the biological function of these structures in cells.

A Pharmacological Chaperone Molecule Induces Cancer Cell Death by Restoring Tertiary DNA Structures in Mutant hTERT Promoters

“…58 Given that the 44-nt 5-12 region contains eight G-rich tracts, multiple G-quadruplex populations can exist, each requiring a minimum of four G-rich tracts. Together with partially folded structures, 42 this constitutes a rather complex array of observable structures in single-molecule mechanical unfolding experiments. Using an approach we have recently established to follow the population dynamics of individual DNA secondary structures with the statistical method PoDNano (Population Deconvolution at Nanometer resolution), 58,70 we first identified the size of different populations (measured in change in contour length [ΔL]; see We designed a medium-throughput assay based on a well-established FRET assay 71 to screen for compounds that might act as chaperones at the early stage of the folding process to shift the population species of the mutants in the 5-12 G-quadruplex back to the larger species.…”

“…shown to be important in the early folding process, 42 this seemed like an attractive postulate to explain the effect of these mutations on hTERT activation. Indeed, both the final folded form and the relative populations of intermediates were severely compromised relative to the WT species.…”

“…Thus we propose that GTC365 can act as a chaperone in both accelerating the folding of the WT and steering the cooperative folding of a mutated 5-12 G-quadruplex to a functional transcriptional repressor (Figure 8; A to B in the WT and C to E to B in the mutants). We have previously shown that the cooperative refolding of the 5-12 G-quadruplex requires the simultaneous folding of the hairpin and the associated G-quadruplex, 42 which is due to tertiary interactions between the hairpin and G-quadruplex structure. Since these interactions occur at early intermediates while the energy barriers may be small, the intermolecular interactions between, for example, the guanidinium group and the 15-mer are still able to drive the secondary structural transitions.…”

“…We have already shown that the tertiary interaction between the hairpin loop and the G-quadruplex results in a folded structure that is stable by 6 kcal/mol energy. 42 Thus, G146 may play a key role in positioning the hairpin loop closer to a loop in the associated G-quadruplex and facilitate this tertiary interaction. Therefore, loss of either interaction results in the formation of a misfolded final structure, which is energetically less stable.…”

“…This structure is important in the formation of the correctly folded structure and transcription silencing ( Figure 1D). 42 An alternative structure that lacks this hairpin loop has been proposed. 43 Significantly, the mouse TERT lacks these 12 G-tracts and has a 16-fold higher transcription activation level.…”