DNA Repair in Real Time: Ultrafast and Single Molecule Studies

实时 DNA 修复：超快单分子研究

• 批准号: 0347087
• 负责人: Robert Stanley
• 金额: $ 44.6万
• 依托单位: Temple University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-03-01 至 2007-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0347087&HistoricalAwards=false
• 关键词: DNA; Repair; Real; Time; Ultrafast

Exposure to ultraviolet radiation (UV) is known to cause DNA damage. The initial insult is often a crosslinking of adjacent nucleic acid bases in DNA generating cyclobutylpyrimidine dimers, or CPDs. All organisms use DNA repair proteins to undo this damaging modification. The project concerns one member of this class of repair proteins, DNA photolyase. Photolyase uses blue light to undo the UV-induced crosslinking. The protein has evolved to identify and bind CPDs with exquisite specificity. Once bound, the CPD is repaired within about 2 billionths of a second upon absorbing blue light. The investigator will use state-of-the-art biophysical and biochemical methods to understand the details of the repair mechanism at the molecular level. These methods include subpicosecond ultrafast laser spectroscopy, both transient absorption and time-resolved fluorescence measurements, single molecule spectroscopy using multiphoton excitation, enzymology, and the development of sophisticated computer models to interpret the results. The repair reaction will be followed in real time, with subpicosecond (less than a millionth of a millionth of a second) time resolution. Other experiments, performed on single protein molecules, will determine how photolyase binds and manipulates the damaged DNA strand. Much of the sophisticated work will be performed by graduate students and post-graduate scholars. Other important experiments will be performed by a cadre of talented undergraduate students. These students will gain a breadth of experience that is a hallmark of biophysical chemistry.

众所周知，暴露于紫外线辐射（UV）会导致DNA损伤。最初的损伤通常是DNA中相邻核酸碱基的交联，产生环丁基嘧啶二聚体或CPDs。所有生物体都使用DNA修复蛋白来消除这种破坏性的修饰。该项目涉及这类修复蛋白的一个成员，DNA光解酶。光解酶使用蓝光解除紫外线诱导的交联。该蛋白已经进化到能够识别和结合cpd，具有非常高的特异性。一旦绑定，CPD在吸收蓝光后，在大约20亿分之一秒内修复。研究者将使用最先进的生物物理和生化方法来了解分子水平上修复机制的细节。这些方法包括亚皮秒超快激光光谱学，瞬态吸收和时间分辨荧光测量，使用多光子激发的单分子光谱学，酶学，以及开发复杂的计算机模型来解释结果。修复反应将实时跟踪，时间分辨率为亚皮秒（小于百万分之一秒）。在单个蛋白质分子上进行的其他实验将确定光解酶如何结合并操纵受损的DNA链。许多复杂的工作将由研究生和研究生学者来完成。其他重要实验将由一群有才华的本科生干部进行。这些学生将获得广泛的经验，这是生物物理化学的标志。