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，可以确定一种或多种中间体的形成，并研究从酶与DNA相互作用之前的瞬间到酶产物释放的过程动力学，每次一个分子。稳态荧光和其他在以前的DNA碱基翻转研究中使用的集合平均技术提供了大量分子平均状态的信息。集合平均测量可以掩盖中间酶-底物复合物形成的波动，并导致对酶促过程的不同解释。在DNA碱基翻转的领域，如果酶将核苷酸“推”出螺旋（主动机制），或者如果酶与临时翻转的碱基结合（被动机制），它仍然有待回答。需要新的单分子方法来全面评估碱翻转过程的动力学机制。紫外线照射会导致DNA内的致癌物质损伤，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。T4末端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处于复合体中时，5'互补腺嘌呤被翻转，结合损伤部位。长期目标是在分子水平上了解酶是如何发现损伤的，以及碱基翻转是如何、何时以及为什么发生的，以修复受损的DNA。这项工作的目标是充分了解T4末端V的修复机制，并确定可以最大限度地修复过程的条件（盐，pH等）。对DNA碱基的破坏会导致突变并导致细胞死亡。例如，如果设计用于修复这些损伤的系统失败，紫外线照射可能导致可能阻止复制的突变。然而，生物体有修复DNA的酶，其中许多酶执行碱基翻转过程来识别、获取和修复受损的核苷酸。这个项目将通过观察单分子荧光来研究这个过程是如何工作的，而不是使用大规模的集合方法。该项目将在西班牙裔服务机构进行，参与该项目的学生将有机会学习各种连接分子生物学，生物化学和物理化学领域的技术。这些技术越来越多地应用于生物物理研究的许多领域，这些经验将激励学生继续在这一领域和其他相关领域进行研究生学习。

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.