RUI: Characterizing DNA target search using single molecule methods

这个项目将研究蛋白质如何在DNA中找到特定的位置。由于人类基因组中有30多亿个碱基对，这简直是大海捞针。将使用多种方法来研究蛋白质如何将滑动、希望和跳跃结合到一种搜索策略中。在一种方法中，将测量蛋白质找到其目标位置所需的时间。在另一种情况下，搜索过程中单个蛋白质的运动将被实时跟踪。在搜索过程中拉伸或卷曲DNA将展示细胞中DNA的不同构象如何影响搜索。通过改变含有DNA和蛋白质的溶液的组成，可以确定不同细胞成分的影响。在另一项调查中，将研究所谓的“非目标”位点(与目标位点相似，通常只有一到两个碱基对差异的位点)如何减缓甚至可能加快搜索速度。实验室工作将由本科生进行，这项研究的概念将包括在首席调查员教授的课程中。此外，研究小组还将为波士顿的中小学生举办夏令营，让这些学生接触研究和科学方法。这个项目将利用单分子方法研究DNA结合蛋白的搜索策略。所选择的模型系统，限制性内切酶，形成细菌先天免疫反应的一部分，并在特定位置切割长度为4-8个碱基对的外源DNA。在一项技术中，与DNA捆绑在一起的微珠将被用来测量单个DNA分子切割的准确时间。使用这种方法，可以在一次实验中测量数百个DNA，以产生高分辨率的动力学数据。第二种技术将使用荧光来跟踪搜索过程中单个蛋白质的运动。一个研究领域将描述滑行、跳跃和跳跃的作用。DNA路障的使用将区分滑动和跳跃，前者将被阻止，后者可以绕过路障。在单分子成像过程中改变DNA的构象可以测试在缠绕过程中是否存在增强的跳跃。另一个领域涉及非靶标结合位点，它可以减缓靶标搜索的速度，但也可以作为库，在转录因子的遗传调控中发挥作用。通过在DNA中包含恒星站点(不同于单个碱基对的非同源站点)，可以测试此类站点的位置和数量如何影响搜索率的定量模型。最后一个领域涉及到大分子拥挤的作用。通过引入拥挤剂，既可以解决目标搜索率(在珠链实验中)的影响，也可以解决对DNA沿线扩散(在单分子荧光跟踪中)的影响。该项目的广泛影响包括对本科生的培训，为本科生课程开发系留DNA模块，以及为中小学生举办夏令营。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

This project will investigate how proteins find specific locations in DNA. Since there are over three billion base pairs in the human genome, this is akin to finding a needle in a haystack. A variety of methods will be employed to investigate how proteins combine sliding, hoping and jumping along DNA into a search strategy. In one method, the time taken by the protein to find its target site will be measured. In another, the motion of individual proteins during search will be tracked in real time. Stretching or coiling the DNA during the search process will demonstrate how the different conformations of DNA in the cell affect the search. By varying the composition of the solution containing DNA and proteins, the effects of different cellular components can be determined. In another investigation, how so called "off-target" sites (sites that are similar to the target site and typically only vary by one or two base pairs) slow or perhaps even speed up the search will be studied. The laboratory work will be carried out by undergraduate students and concepts from this research will be included in courses taught by the principal investigator. In addition, the research team will hold summer day camps for students from elementary and middle schools in Boston, where these students will be exposed to research and the scientific method.This project will investigate search strategies of DNA binding proteins using single molecule methods. The model system chosen, restriction endonucleases, form part of the bacterial innate immune response and cleave foreign DNA at specific sites 4-8 base pairs in length. In one technique, micro beads tethered with DNA will be used to measure the exact time of cleavage of individual DNA molecules. Using this method, hundreds of DNAs can be measured in a single experiment to yield high resolution kinetic data. A second technique will use fluorescence to track the motion of individual proteins during search. One area of investigation will characterize the roles of sliding, hopping and jumping. The use of DNA roadblocks will distinguish between sliding, which will be blocked, and hopping, which can bypass roadblocks. Varying the conformation of DNA during single molecule imaging can test for the presence of enhanced jumping during coiling. Another area concerns off-target binding sites, which can slow target search, but also serve as reservoirs that can play a role in genetic regulation by transcription factors. By inclusion of star sites (non-cognate sites differing by a single base pair) in DNA, quantitative models of how the position and number of such sites affect search rates can be tested. A final area involves the role of macromolecular crowding. By introducing crowding agents, the effects both on target search rates (in bead tethering experiments) and on linear diffusion along DNA (in single molecule fluorescence tracking) can be addressed. Broader impacts of this project include training of undergraduate students, developing modules on tethered DNA for undergraduate courses, as well as a summer day camp for elementary and middle school students.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.