4 research outputs found
A Mechanosensor Mechanism Controls the G‑Quadruplex/i-Motif Molecular Switch in the <i>MYC</i> Promoter NHE III<sub>1</sub>
<i>MYC</i> is overexpressed in many different cancer
types and is an intensively studied oncogene because of its contributions
to tumorigenesis. The regulation of <i>MYC</i> is complex,
and the NHE III<sub>1</sub> and FUSE elements rely upon noncanonical
DNA structures and transcriptionally induced negative superhelicity.
In the NHE III<sub>1</sub> only the G-quadruplex has been extensively
studied, whereas the role of the i-motif, formed on the opposite C-rich
strand, is much less understood. We demonstrate here that the i-motif
is formed within the 4CT element and is recognized by hnRNP K, which
leads to a low level of transcription activation. For maximal hnRNP
K transcription activation, two additional cytosine runs, located
seven bases downstream of the i-motif-forming region, are also required.
To access these additional runs of cytosine, increased negative superhelicity
is necessary, which leads to a thermodynamically stable complex between
hnRNP K and the unfolded i-motif. We also demonstrate mutual exclusivity
between the <i>MYC</i> G-quadruplex and i-motif, providing
a rationale for a molecular switch mechanism driven by SP1-induced
negative superhelicity, where relative hnRNP K and nucleolin expression
shifts the equilibrium to the on or off state
Mutually Exclusive Formation of G‑Quadruplex and i‑Motif Is a General Phenomenon Governed by Steric Hindrance in Duplex DNA
G-Quadruplex
and i-motif are tetraplex structures that may form
in opposite strands at the same location of a duplex DNA. Recent discoveries
have indicated that the two tetraplex structures can have conflicting
biological activities, which poses a challenge for cells to coordinate.
Here, by performing innovative population analysis on mechanical unfolding
profiles of tetraplex structures in double-stranded DNA, we found
that formations of G-quadruplex and i-motif in the two complementary
strands are mutually exclusive in a variety of DNA templates, which
include human telomere and promoter fragments of hINS and hTERT genes.
To explain this behavior, we placed G-quadruplex- and i-motif-hosting
sequences in an offset fashion in the two complementary telomeric
DNA strands. We found simultaneous formation of the G-quadruplex and
i-motif in opposite strands, suggesting that mutual exclusivity between
the two tetraplexes is controlled by steric hindrance. This conclusion
was corroborated in the BCL-2 promoter sequence, in which simultaneous
formation of two tetraplexes was observed due to possible offset arrangements
between G-quadruplex and i-motif in opposite strands. The mutual exclusivity
revealed here sets a molecular basis for cells to efficiently coordinate
opposite biological activities of G-quadruplex and i-motif at the
same dsDNA location
Click Chemistry Assisted Single-Molecule Fingerprinting Reveals a 3D Biomolecular Folding Funnel
A 3D folding funnel was proposed in the 1990s to explain
the fast
kinetics exhibited by a biomacromolecule in presence of seemingly
unlimited folding pathways. Over the years, numerous simulations have
been performed with this concept; however, experimental verification
is yet to be attained even for the simplest proteins. Here, we have
used a click chemistry based strategy to introduce six pairs of handles
in a human telomeric DNA sequence. A laser-tweezers-based, single-molecule
structural fingerprinting on the six inter-handle distances reveals
the formation of a hybrid-1 G-quadruplex in the sequence. Kinetic
and thermodynamic fingerprinting on the six trajectories defined by
each handle-pair depict a 3D folding funnel and a kinetic topology
in which the kinetics pertaining to each handle residue is annotated
for this G-quadruplex. We anticipate the methods and the concepts
developed here are well applicable to other biomacromolecules, including
RNA and proteins
Tertiary DNA Structure in the Single-Stranded hTERT Promoter Fragment Unfolds and Refolds by Parallel Pathways via Cooperative or Sequential Events
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 also this unexpected complexity in a promoter element provides
additional justification for the biological function of these structures
in cells