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，用于确定一个或多个中间体的形成，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的动力学，每次一个分子。在以前的DNA碱基翻转研究中使用的稳态荧光和其他系综平均技术提供了大量分子的平均状态的信息。包被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碱基翻转领域，如果酶将核苷酸“推出”螺旋（主动机制）或如果酶结合到临时翻转的碱基（被动机制），仍然有待回答。需要新的单分子方法来充分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内切核酸酶V（T4 endo V）是一种细菌DNA修复酶，可以消除CPD。T4 endo V的晶体结构显示，当该酶与含有胸腺嘧啶二聚体的螺旋DNA形成复合物时，5'互补腺嘌呤被翻转出来，结合损伤位点。长期目标是在分子水平上了解酶如何发现损伤，以及如何，何时以及为什么碱基翻转发生以修复受损的DNA。这项工作的目标是充分了解T4 endo V的修复机制，并确定条件（盐，pH值等）。可以最大化修复过程。DNA碱基的损伤会导致突变并导致细胞死亡。例如，紫外线照射可能导致突变，如果设计用于修复这些损伤的系统失败，则可能会阻止复制。然而，生物体具有修复DNA的酶，并且这些酶中的许多执行碱基翻转过程以识别、获得和修复受损的核苷酸。该项目将通过观察单分子荧光而不是使用大规模集成方法来研究这一过程是如何工作的。该项目将在西班牙裔服务机构进行，参加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。许多这些技术越来越多地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个和其他相关领域的研究生学习。

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.