Replication protein A unfolds G-quadruplex structures with varying degrees of efficiency.
Replication protein A unfolds G-quadruplex structures with varying degrees of efficiency.
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DOI：
10.1021/jp300546u
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发表时间：
2012-05-17
期刊：
影响因子：
--
通讯作者：
Balci H
中科院分区：
文献类型：
--
作者：
Qureshi MH;Ray S;Sewell AL;Basu S;Balci H
已知复制蛋白A （RPA）与采用g -四重体（GQ）结构的富含g的序列相互作用。文献中的大多数研究都是对同源序列（如人类端粒重复序列）形成的GQ进行的，而RPA揭示不同稳定性GQ结构的能力尚不清楚。我们通过单分子FRET和大量生物物理和生化实验，比较了三种潜在的GQ形成DNA序列（PQS）的热稳定性及其对RPA介导的展开的稳定性。其中一个序列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的序列是高度异质的序列，较好地代表了基因组中的PQS。三种GQ结构体的热稳定性有显著差异。我们的测量表明，最热稳定的结构（Tm= 86°C）对RPA介导的展开也是最稳定的，尽管最不热稳定的结构（Tm= 69°C）对RPA介导的展开的稳定性至少比具有中等热稳定性的结构（Tm= 78°C）高一个数量级。当考虑到细胞环境中蛋白质- dna相互作用可能是GQ生存能力的重要决定因素时，这一观察结果的意义变得更加明显。考虑到这些，我们得出结论，热稳定性不一定是预测GQ结构生理活力的适当标准。最后，我们使用单分子止流方法测量了RPA分子将GQ从完全折叠到完全展开的构象所需的时间。所有三个GQ结构都在Δt≈0.30±0.10秒内展开，这是一个令人惊讶的结果，因为展开时间与热稳定性或抗RPA介导的展开稳定性无关。这些结果表明，RPA对g -四重体展开的限制步骤仅仅是结构对RPA蛋白的可及性。
Replication Protein A (RPA) is known to interact with G-rich sequences that adopt G-quadruplex (GQ) structures. Most studies in the literature have been performed on GQ formed by homogenous 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 (PQS) to their stability against RPA mediated unfolding using single molecule 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, which better represent PQS in the genome. The three GQ constructs have thermal stabilities that are significantly different from each other. 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 compared to the structure with intermediate thermal stability (Tm= 78 °C). The significance of this observation becomes more evident when considered within the context of cellular environment where protein-DNA interactions can be an important determinant of GQ viability. Considering these, we conclude that thermal stability is not necessarily an adequate criterion for predicting 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 type method. All three GQ structures were unfolded within Δt≈0.30±0.10 sec, a surprising result as 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.
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