Mechanism of protein-mediated looping in a complex environment

描述（由申请人提供）：细胞中DNA的拓扑结构和空间组织对其功能至关重要，并受到蛋白质的严格调控。蛋白质介导结构的两个突出特征是DNA环和凝聚DNA的形成。环允许蛋白质-蛋白质和/或蛋白质-DNA在大的基因组距离上相互作用。蛋白质介导的DNA环的一个例子是端粒结构。端粒是一种核蛋白结构，覆盖在线性染色体的末端。人类端粒DNA可以排列成数千个碱基对大小的DNA T环。只有静态快照的T-环的形成是已知的。TRF 1和TRF 2是与环形成相关的必需蛋白，但它们在T环形成动力学中的确切作用和功能尚不清楚。 核DNA存在于固有的拥挤环境中，其中含有非常高体积分数的DNA和蛋白质。一般来说，存在有序域的层次结构，因此传统的拥挤模型可能无法完全捕获环境的影响。 为了提高对循环过程的理解，我们建议通过将DNA和蛋白质限制在纳米流体体积中来研究单分子水平上的时间分辨DNA循环。我们认为，这种限制模仿某些方面的核环境，而不是复制拥挤的小分子。我们将使用纳米通道限制的端粒DNA观察TRF 1和TRF 2的动态行为，并形成端粒环如何形成的操作模型。对模型酶T4连接酶进行相同的操作。所有的实验都使用真实的动态单分子。 为了建立限制在环形成中的作用，我们将应用高分辨率AFM成像来推导T4连接酶、TRF 1和TRF 2介导的DNA环在纳米通道内形成的静态构象以及在自由溶液中形成的静态构象。与由模型系统T4 DNA连接酶形成的DNA环的比较将有助于解释通过TRF 1/TRF 2介导的成环机制。 在最后一项研究中，我们将在纳米通道结中相互平行或垂直排列独立的DNA链，并使用荧光成像来测量连接酶和TRF 1/TRF 2的捕获率，以真实的时间跟踪DNA分子相互扫描通过的动作。我们预计，与单纯的点接触相比，平行移动的分子彼此经过会增加DNA-DNA捕获。 这些结果对于推进我们对端粒生物学的理解、几何约束对核DNA利用率的影响以及受限的刚性生物聚合物的物理特性都是至关重要的。

DESCRIPTION (provided by applicant): The topology and spatial organization of DNA in cells is essential to its function and is tightly regulated by proteins. Two prominent features of protei-mediated structure are DNA loops and the formation of condensed DNA. Looping allows protein-protein and/or protein-DNA interactions over large genomic distances. An example of protein-mediated DNA looping is the telomere structure. Telomeres are nucleoprotein structures that cap the ends of linear chromosomes. Human telomeric DNA can be arranged into DNA T-loops as large as thousands of base pairs. Only static snapshots of T-loop formation are known. TRF1 and TRF2 are essential proteins associated with the loop formation, but their precise role and function in the dynamics of T-loop formation remains unclear. Nuclear DNA exists in an intrinsically crowded environment containing a very high volume fraction of DNA and proteins. Generally there is a hierarchy of domains of order, and thus the traditional crowding model may not fully capture the influence of the environment. To improve understanding of the looping process, we propose to study time-resolved DNA looping at the single-molecule level by confining DNA and proteins to nanofluidic volume. We argue that this confinement mimics certain aspects of the nuclear environment that are not reproduced by crowding in small molecules. We will use nanochannel-confined telomeric DNA to observe the dynamic actions of TRF1 and TRF2, and form an operational model of how the telomeric loop forms. The same will be performed for the model enzyme T4 ligase. All experiments use dynamic single molecules in real time. To establish the role of confinement in loop formation, we will apply high-resolution AFM imaging to derive the static conformations of T4 ligase, TRF1 and TRF2 mediated DNA loops formed inside nanochannels and that formed in free solution. Comparison with DNA loops formed by the model system, T4 DNA ligase, will aid in the interpretation of looping mechanisms mediated through TRF1/TRF2. In the last study, we will align independent DNA strands parallel or perpendicular to each other in nanochannel junctions, and follow the actions in real time using fluorescence imaging to measure the capture rate by ligase and TRF1/TRF2 as DNA molecules are scanned past each other. We anticipate that moving molecules in parallel past each other increases DNA-DNA capture when compared to mere point contacts. The results are essential for advancing our understanding of telomere biology, the influence of geometric constraints on the utilization of nuclear DNA, and physics of confined stiff biopolymers in general.