Department of Physics, †Department of Biological Sciences, and ∥Department of Chemistry and Biochemistry, Kent State University , Kent, Ohio 44242, United States.
J Phys Chem B. 2012 May 17;116(19):5588-94. doi: 10.1021/jp300546u. Epub 2012 May 3.
Replication 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.
复制蛋白 A（RPA）已知与富含鸟嘌呤（G）的序列相互作用，这些序列会形成 G-四链体（GQ）结构。文献中报道的大多数研究都是在均一序列形成的 GQ 上进行的，例如人类端粒重复序列，而 RPA 展开不同稳定性的 GQ 结构的能力尚不清楚。我们使用单分子荧光共振能量转移（FRET）和体生物物理和生化实验比较了三个潜在的 GQ 形成 DNA 序列（PQS）的热稳定性与其对 RPA 介导的展开的稳定性。这些序列之一是人类端粒重复序列，另外两个位于酪氨酸羟化酶基因启动子区域，是高度异质的序列，它们更能代表基因组中的 PQS。三个 GQ 结构的热稳定性有显著差异。我们的测量结果表明，最稳定的结构（Tm = 86°C）也是最能抵抗 RPA 介导的展开的结构，尽管最不稳定的结构（Tm = 69°C）对 RPA 介导的展开的稳定性至少比具有中间热稳定性的结构（Tm = 78°C）高一个数量级。当考虑到蛋白质-DNA 相互作用可能是 GQ 存活的重要决定因素的细胞环境时，这种观察结果的意义变得更加明显。考虑到这些结果，我们得出结论，热稳定性不一定是预测 GQ 结构生理可行性的充分标准。最后，我们使用单分子停流方法测量了 RPA 分子将 GQ 从完全折叠状态展开到完全展开状态所需的时间。所有三个 GQ 结构都在 Δt ≈ 0.30 ± 0.10 s 内展开，这是一个令人惊讶的结果，因为展开时间与热稳定性或对 RPA 介导的展开的稳定性无关。这些结果表明，RPA 展开 GQ 的限制步骤仅是结构对 RPA 蛋白的可及性。
