Kinetic partitioning modulates human telomere DNA G-quadruplex structural polymorphism.

Telomeres are specialized chromatin structures found at the end of chromosomes and are crucial to the maintenance of eukaryotic genome stability. Human telomere DNA is comprised of the repeating sequence (T2AG3)n, which is predominantly double-stranded but terminates with a 3’ single-stranded tail. The guanine-rich tail can fold into secondary structures known as a G-quadruplexes (GQs) that may exist as a polymorphic mixture of anti-parallel, parallel, and several hybrid topological isomers. Using single-molecule Förster resonance energy transfer (smFRET), we have reconstructed distributions of telomere DNA GQ conformations generated by an in situ refolding protocol commonly employed in single-molecule studies of GQ structure, or using a slow cooling DNA annealing protocol typically used in the preparation of GQ samples for ensemble biophysical analyses. We find the choice of GQ folding protocol has a marked impact on the observed distributions of DNA conformations under otherwise identical buffer conditions. A detailed analysis of the kinetics of GQ folding over timescales ranging from minutes to hours revealed the distribution of GQ structures generated by in situ refolding gradually equilibrates to resemble the distribution generated by the slow cooling DNA annealing protocol. Interestingly, conditions of low ionic strength, which promote transient GQ unfolding, permit the fraction of folded DNA molecules to partition into a distribution that more closely approximates the thermodynamic folding equilibrium. Our results are consistent with a model in which kinetic partitioning occurs during in situ folding at room temperature in the presence of K+ ions, producing a long-lived non-equilibrium distribution of GQ structures in which the parallel conformation predominates on the timescale of minutes. These results suggest that telomere DNA GQ folding kinetics, and not just thermodynamic stability, likely contributes to the physiological ensemble GQ structures.

端粒是位于染色体末端的特殊染色质结构，对维持真核生物基因组的稳定性至关重要。人类端粒DNA由重复序列(T2AG3)n组成，其主要是双链的，但以3’单链尾巴终止。富含鸟嘌呤的尾巴可以折叠成称为G - 四链体（GQs）的二级结构，它可能以反平行、平行以及几种杂合拓扑异构体的多态混合物形式存在。利用单分子福斯特共振能量转移（smFRET），我们重构了由单分子GQ结构研究中常用的原位重折叠方案产生的端粒DNA GQ构象分布，或者使用在制备用于整体生物物理分析的GQ样本时通常使用的缓慢冷却DNA退火方案产生的构象分布。我们发现GQ折叠方案的选择在其他条件相同的缓冲液条件下对观察到的DNA构象分布有显著影响。对从几分钟到几小时时间尺度上的GQ折叠动力学的详细分析表明，原位重折叠产生的GQ结构分布逐渐平衡，变得类似于缓慢冷却DNA退火方案产生的分布。有趣的是，低离子强度条件促进GQ短暂解折叠，使得折叠的DNA分子比例分配成一种更接近热力学折叠平衡的分布。我们的结果与一个模型相符，在该模型中，在室温下有K⁺离子存在时的原位折叠过程中发生动力学分配，产生一种GQ结构的长期非平衡分布，其中平行构象在几分钟的时间尺度上占主导。这些结果表明，端粒DNA GQ折叠动力学，而不仅仅是热力学稳定性，可能对生理上的GQ结构整体有贡献。