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Replication Protein A Unfolds G-Quadruplex Structures with Varying Degrees of Efficiency
journal contribution
posted on 2012-05-17, 00:00 authored by Mohammad
H. Qureshi, Sujay Ray, Abby L. Sewell, Soumitra Basu, Hamza BalciReplication protein A (RPA) is known to interact with
guanine-
(G-) rich sequences that adopt G-quadruplex (GQ) structures. Most
studies reported in the literature were performed on GQ formed by
homogeneous sequences, such as the human telomeric repeat, and RPA’s
ability to unfold GQ structures of differing stability is not known.
We compared the thermal stability of three potential GQ-forming DNA
sequences (PQSs) to their stability against RPA-mediated unfolding
using single-molecule fluorescence resonance energy transfer (FRET)
and bulk biophysical and biochemical experiments. One of these sequences
is the human telomeric repeat and the other two, located in the promoter
region of tyrosine hydroxylase gene, are highly heterogeneous sequences
that better represent PQSs in the genome. The three GQ constructs
have thermal stabilities that differ significantly. Our measurements
showed that the most thermally stable structure (Tm = 86 °C) was also the most stable against RPA-mediated
unfolding, although the least thermally stable structure (Tm = 69 °C) had at least an order-of-magnitude
higher stability against RPA-mediated unfolding than the structure
with intermediate thermal stability (Tm = 78 °C). The significance of this observation becomes more
evident when considered within the context of the cellular environment
where protein–DNA interactions can be an important determinant
of GQ viability. Considering these results, we conclude that thermal
stability is not necessarily an adequate criterion for predicting
the physiological viability of GQ structures. Finally, we measured
the time it takes for an RPA molecule to unfold a GQ from a fully
folded to a fully unfolded conformation using a single-molecule stopped-flow
method. All three GQ structures were unfolded within Δt ≈ 0.30 ± 0.10 s, a surprising result considering
that the unfolding time does not correlate with thermal stability
or stability against RPA-mediated unfolding. These results suggest
that the limiting step in G-quadruplex unfolding by RPA is simply
the accessibility of the structure to the RPA protein.