Single-molecule investigations of fast biomolecular search processes initiating DNA repair

我的研究计划的总体目标是建立一个新的机制的快速分子搜索过程的理解，这是一个主机的关键细胞过程的基础。一类快速分子搜索过程，由于有限的分析技术，在很大程度上仍然没有表征，是在电离辐射、自由基和复制期间切割DNA后启动DNA修复的恒定过程的搜索。 现有的显微镜技术是不能够表征这些分子的搜索在生理条件下，因为它们不能同时解决：弱蛋白质-DNA和DNA-DNA相互作用;延长搜索轨迹的个别分子在几秒钟内，和毫秒的时间尺度的相互作用动力学;也不是他们与拓扑结构复杂或应变的DNA基板兼容。我将使用一套新的定制单显微镜技术来规避这些限制，这些技术扩展了CLIC成像。 我的第一个短期目标是创建一个独特的体外显微镜工作站，能够解决有关由校准良好的MRX修复蛋白复合物进行的分子搜索的开放性问题，该复合物的生物物理特性和DNA相互作用以前已经测量过，但条件范围有限。我的第二个短期目标是应用这些技术对9-1-1修复蛋白复合物进行首次单分子研究，该复合物仅通过生物化学，细胞生物学和遗传分析进行研究。我的长期目标是扩展CLIC成像，以创建一个独特的力和荧光体外显微镜站，我将适用于探测分子搜索的DNA基板的控制拓扑结构和施加的力。该研究计划为在系统地接近复杂细胞环境的条件下对广泛的分子搜索过程（例如信使RNA的转录和翻译）进行首次单分子研究奠定了基础，并在生物医学研究中有无数应用。

The overarching objective of my research program is to create a new mechanistic understanding of the fast molecular search processes, which underlie a host of key cellular processes. One class of fast molecular search processes, that remain largely uncharacterized due to limited analysis techniques, is the search that initiates the constant processes of DNA repair following its cleavage by ionizing radiation, free radicals and during replication. Existing microscopy techniques are not capable of characterizing these molecular searches under physiological conditions because they can not simultaneously resolve: weak protein-DNA and DNA-DNA interactions; extended search trajectories of individual molecules over several seconds, and millisecond-timescale interaction kinetics; nor are they compatible with topologically complex or strained DNA substrates. I will circumvent these limitations using a new suite of custom single-microscopy techniques which extend CLIC-imaging. My first short-term objective is to create a unique in vitro microscopy station, capable of addressing open questions about the molecular searches performed by the well-calibrated MRX repair protein complex, whose biophysical properties and DNA-interactions have been measured previously, though under a limited range of conditions. My second short-term objective is to apply these techniques to perform the first single-molecule investigations of the 9-1-1 repair protein complex, which has only been studied with biochemical, cell-biological and genetic assays. My long-term objective is to extend CLIC-imaging to create a unique force and fluorescence in vitro microscopy station, which I will apply to probe molecular searches on DNA substrates of controlled topology and applied force. This research program sets the stage for the first single-molecule investigations of a wide range of molecular search processes (e.g. transcription and translation of messenger RNA) under conditions that systematically approach the complex cellular milieu, with myriad applications to biomedical research.