Time-Resolved Photochemical Footprinting

9410748已研究并测试了Brenowitz光化学技术，使蛋白质-DNA复合物的结构的时间分辨测定成为可能，从而可以检查反应中间体。 本项目将1）确定光解钴离子切割DNA的机制和特异性; 2）开发快速混合激光光解方法，以实现光化学足迹的毫秒时间分辨率的目标; 3）通过检测两种序列特异性DNA结合蛋白的反应机制来测试新技术。 这种新技术将是非常有价值的，在弥合目前的热力学和结构分析的蛋白质-DNA相互作用的差距。 由于复合物的形成可以在单个碱基对分辨率下进行，因此详细的结构信息将在毫秒至微秒的时间分辨率下可用。 将使用EcoRI限制性内切酶与其识别序列的结合以及“TATA”结合蛋白（TBP）与启动子的结合来测试光化学足迹方案。 因此，有可能首次研究蛋白质-核酸复合物形成的中间步骤。 了解蛋白质识别和结合特定DNA序列的机制是了解细胞功能和功能障碍的关键一步。 蛋白质-DNA相互作用在基因复制、重组和调控等细胞过程中起着重要作用。 蛋白质DNA复合物的形成是一个复杂的过程，涉及多个步骤和许多不同的化学相互作用。 通过使用确定复合物的三维结构的技术，以及独立地通过探测相互作用的化学性质的技术，已经对蛋白质与特定DNA序列结合的机制有了重要的见解。 然而，这些数据的一个严重局限性是它们仅报告结合反应的终点。 因此，结合反应中的中间事件，如初始识别位点的鉴定和DNA构象变化的诱导尚未被观察或研究。 正在开发利用可见光引发化学反应的技术，这将使蛋白质与特定DNA序列反应的时间依赖性得以检查。 这种方法将大大推进我们对蛋白质识别和结合DNA特定序列的分子机制的理解。 ***

9410748 Brenowitz Photochemical techniques have been researched and tested enabling time-resolved determinations of the structures of protein-DNA complexes that allow the reaction intermediates to be examined. This project will 1) Determine the mechanism and specificity of cleavage of DNA by photolyzed cobalamins; 2) Develop a rapid mixing-laser photolysis method to attain the goal of millisecond time resolution for photochemical footprinting; and 3) Test the new technology by examining the reaction mechanism of two sequence- specific DNA binding proteins. This new technique will be immensely valuable in bridging the gaps in present thermodynamic and structural analysis of protein-DNA interactions. Since complex formation can be followed at single basepair resolution, detailed structural information will be available at time resolutions on the order of milliseconds to microseconds. The photochemical footprinting protocols will be tested using the binding of the Eco RI restriction endonuclease to its recognition sequence and the binding of the 'TATA' Binding Protein (TBP) to a promoter. Thus, it will be possible to study for the first time the intermediate steps in the formation of the protein-nucleic acid complex. %%% Understanding the mechanisms by which proteins recognize and bind to specific DNA sequences is a key step towards understanding cellular function and dysfunction. Protein-DNA interactions play important roles in cellular processes such as gene replication, recombination and regulation. The formation of protein DNA complexes is a complex process involving multiple steps and many different chemical interactions. Significant insights have been made into the mechanisms by which proteins bind to specific DNA sequences using techniques that determine the three-dimensional structure of the complexes, and independently by techniques that probe the chemistry of the interactions. However, a severe limitation of such data is that they report only on the endpoints of the binding reaction. Thus, the intermediate events in the binding reaction, such as identification of initial recognition sites and induction of changes in the conformation of the DNA have not been observed or studied. Techniques are under development that utilize visible light to trigger chemical reactions that will enable the time dependence of the reaction of proteins with specific DNA sequences to be examined. This approach will greatly advance our understanding of the molecular mechanism by which proteins recognize and bind to specific sequences of DNA. ***