Research Starter Grant: Single Molecule Fluorescence Study of the DNA Repair Mechanism of T4 Endonuclease V

单分子荧光(SMF)是一种强大的技术，可以确定一个或多个中间体的形成，并研究从酶与DNA相互作用前的瞬间到酶产物释放的过程的动力学，一次一个分子。在以前的DNA碱基翻转研究中使用的稳态荧光和其他集合平均技术提供了关于大量分子的平均状态的信息。集合平均测量可以掩盖中间酶-底物复合体形成的波动，并导致对酶过程的不同解释。在DNA碱基翻转方面，如果酶将核苷酸“推”出螺旋结构(主动机制)，或者如果酶结合到临时的翻转碱基(被动机制)，这个问题仍然有待回答。需要新的单分子方法来全面评估碱基翻转过程的动力学机制。紫外线照射会导致DNA内的致癌损伤，包括环丁烷嘧啶二聚体(CPD)的形成，这是最常见的紫外线DNA损伤类型。T4内切酶V(T4 Endo V)是一种消除CPD的细菌DNA修复酶。T4Endo V的晶体结构表明，当该酶与含有胸腺嘧啶二聚体的螺旋DNA形成复合体时，5‘互补腺嘌呤被翻转出来，与损伤部位结合。长期目标是在分子水平上了解酶是如何发现损伤的，以及如何、何时和为什么发生碱基翻转来修复受损的DNA。这项工作的目的是充分了解T4 Endo V的修复机制，并确定条件(盐、pH等)。这可以最大限度地提高修复过程。DNA碱基的破坏可能导致突变并导致细胞死亡。例如，紫外线照射可能导致突变，如果旨在修复这些损伤的系统失败，可能会阻止复制。然而，生物体内有修复DNA的酶，其中许多酶执行碱基翻转过程来识别、获得和修复受损的核苷酸。这个项目将通过观察单分子荧光而不是使用大规模的系综方法来研究这一过程是如何工作的。拟议的项目将在西班牙裔服务机构进行，在那里参与该项目的学生将有机会学习连接分子生物学、生物化学和物理化学领域的各种技术。其中许多技术越来越多地被用于生物物理研究的许多领域，这一经历将激励学生在这个领域和其他相关领域继续研究生学习。

Single-molecule fluorescence (SMF) is a powerful technique to determine the formation of one or more intermediates, and to study the kinetics of the processes from the instant before an enzyme interacts with the DNA until the release of the enzymatic product, one molecule at a time. Steady-state fluorescence and other ensemble average techniques used in previous DNA base flipping studies provide information about the average state of a large number of molecules. Ensemble averaged measurements can mask fluctuations in the formation of intermediate enzyme-substrate complexes and lead to different interpretations of the enzymatic process. In the area of DNA base flipping it still remains to be answered if the enzyme "pushes" the nucleotide out of the helix (active mechanism) or if the enzyme binds to a provisional flipped base (passive mechanism). New single molecule approaches to fully assess the kinetics mechanism of the base flipping process are needed. UV irradiation causes carcinogen-lesions within DNA, including the formation of cyclobutane pyrimidine dimers (CPD), which are the most common type of UV DNA damage. T4 endonuclease V (T4 endo V) is a bacterial DNA repair enzyme that eliminates CPD. The crystal structure of T4 endo V shows that when the enzyme is in a complex with a helical DNA containing a thymine dimer, the 5' complementary adenine is flipped out, binding the damage site. The long-term goal is to understand at the molecular level how the enzyme finds the damage, and how, when, and why the base flipping occurs to repair damaged DNA. The goal of this work is to fully understand the repair mechanism of T4 endo V and to determine the conditions (salt, pH, etc.) that could maximize the repair process. Damage to DNA bases can result in mutations and lead to cell death. For example, UV irradiation can result in mutations that could block replication if the systems designed to repair these damages fail. However, living organisms have enzymes to repair DNA, and many of these enzymes perform a base flipping process to recognize, gain access to, and repair damaged nucleotides. This project will study how this process works by looking at single molecules fluorescence instead of using large-scale ensemble methods. The proposed project will be performed in a Hispanic Service Institution, where the students that will participate in this project will have opportunities to learn a variety of techniques that connect the fields of Molecular Biology, Biochemistry, and Physical Chemistry. Many of these techniques are increasingly used in many areas of biophysical research and the experience will inspire students to continue graduate studies in this and other related fields.