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

研究启动资助：T4 核酸内切酶 V 的 DNA 修复机制的单分子荧光研究

• 批准号: 1237548
• 负责人: Elvin Aleman
• 金额: $ 4.99万
• 依托单位: California State University-Stanislaus
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-07-01 至 2013-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1237548&HistoricalAwards=false
• 关键词: Research; Starter; Grant; Single; Molecule

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.

单分子荧光 (SMF) 是一种强大的技术，可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（一个分子在一次。先前 DNA 碱基翻转研究中使用的稳态荧光和其他整体平均技术提供了有关大量分子平均状态的信息。整体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，仍然有待解答的是酶是否将核苷酸“推出”螺旋（主动机制）或酶是否与临时翻转碱基结合（被动机制）。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 核酸内切酶 V (T4 endo V) 是一种细菌 DNA 修复酶，可消除 CPD。 T4 endo 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 形成复合物时，5' 互补腺嘌呤被翻转出来，与损伤位点结合。长期目标是在分子水平上了解酶如何发现损伤，以及碱基翻转如何、何时以及为何发生以修复受损的 DNA。这项工作的目标是充分了解 T4 endo V 的修复机制，并确定可以最大化修复过程的条件（盐、pH 等）。 DNA 碱基的损伤会导致突变并导致细胞死亡。例如，如果用于修复这些损伤的系统失败，紫外线照射可能会导致突变，从而阻碍复制。然而，生物体具有修复 DNA 的酶，其中许多酶会执行碱基翻转过程来识别、获取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该过程的工作原理。拟议的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该领域和其他相关领域进行研究生学习。