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）是确定一个或多个中间体形成的强大技术，并在酶与DNA相互作用之前从瞬间开始研究过程的动力学，直到酶产物释放到一次分子。先前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有关大量分子的平均状态的信息。合奏平均测量可以掩盖中间酶 - 底物复合物的形成，并导致对酶促过程的不同解释。在DNA碱基翻转的区域中，如果将酶从螺旋中“推出”核苷酸（主动机构）或该酶与临时翻转碱（被动机构）结合，则仍然有待回答。需要新的单分子方法，以充分评估基本翻转过程的动力学机制。紫外线照射会导致DNA内的致癌物，包括形成环丁烷嘧啶二聚体（CPD），这是紫外DNA损伤的最常见类型。 T4核酸内切酶V（T4 Endo V）是一种消除CPD的细菌DNA修复酶。 T4 Endo V的晶体结构表明，当酶与含有胸腺素二聚体的螺旋DNA配合在一起时，5'互补腺嘌呤会翻转，从而结合了损伤位点。长期的目标是在分子水平上了解酶如何找到损伤，以及如何，何时以及为什么发生损坏以修复受损的DNA。这项工作的目的是充分了解T4 Endo V的维修机制，并确定可以最大化维修过程的条件（盐，pH等）。对DNA碱基的损害会导致突变并导致细胞死亡。例如，如果旨在修复这些损害的系统失败的系统，紫外线照射可能会导致突变，从而阻止复制。然而，活生物体具有修复DNA的酶，其中许多酶执行了基础翻转过程，以识别，获取和修复受损的核苷酸。该项目将通过查看单分子荧光而不是使用大型集合方法来研究该过程的工作方式。拟议的项目将在西班牙裔服务机构中进行，在那里将参加该项目的学生将有机会学习各种技术，这些技术将分子生物学，生物化学和物理化学领域连接起来。这些技术中的许多越来越多地用于生物物理研究的许多领域，这种经验将激发学生继续在该和其他相关领域的研究生学习。