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节。蛋白质介导结构的两个突出特征是DNA环和浓缩DNA的形成。环允许蛋白质-蛋白质和/或蛋白质- dna在大基因组距离上相互作用。端粒结构是蛋白质介导的DNA环的一个例子。端粒是覆盖线性染色体末端的核蛋白结构。人类端粒DNA可以排列成数千个碱基对大小的DNA t环。目前只知道t型环形成的静态快照。TRF1和TRF2是与t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存在于本质上拥挤的环境中，其中含有非常高体积分数的DNA和蛋白质。一般来说，拥挤区域是有层次的，因此传统的拥挤模型可能不能完全反映环境的影响。为了提高对环过程的理解，我们建议在单分子水平上通过将DNA和蛋白质限制在纳米流体体积内来研究时间分辨DNA环。我们认为，这种约束模拟了核环境的某些方面，而这些方面是不能通过小分子拥挤来复制的。我们将使用纳米通道限制的端粒DNA来观察TRF1和TRF2的动态作用，并形成端粒环如何形成的操作模型。对模型酶T4连接酶进行同样的处理。所有的实验都是实时使用动态的单分子。为了确定约束在环形成中的作用，我们将应用高分辨率原子力显微镜成像来得出T4连接酶、TRF1和TRF2介导的DNA环在纳米通道内形成和在自由溶液中形成的静态构象。与模型系统（T4 DNA连接酶）形成的DNA环比较，将有助于解释通过TRF1/TRF2介导的环机制。在最后一项研究中，我们将在纳米通道连接处排列彼此平行或垂直的独立DNA链，并使用荧光成像实时跟踪这些动作，以测量DNA分子相互扫描时连接酶和TRF1/TRF2的捕获率。我们预计，与单纯的点接触相比，平行移动分子可以增加DNA-DNA捕获。这些结果对于提高我们对端粒生物学、几何约束对核DNA利用的影响以及一般受限刚性生物聚合物的物理学的理解至关重要。

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.