DNA Repair in Real Time: Ultrafast and Single Molecule Studies

已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CPD。所有生物体都使用DNA修复蛋白来消除这种破坏性的修改。该项目涉及这类修复蛋白的一个成员，DNA光裂合酶。Photolyase使用蓝光来消除UV诱导的交联。这种蛋白质已经进化到能够识别和结合具有精确特异性的CPD。一旦结合，CPD在吸收蓝光后在大约20亿分之一秒内修复。研究人员将使用最先进的生物物理和生物化学方法，在分子水平上了解修复机制的细节。这些方法包括亚皮秒超快激光光谱，瞬态吸收和时间分辨荧光测量，单分子光谱使用多光子激发，酶学，和复杂的计算机模型的发展来解释的结果。修复反应将在真实的时间内进行，时间分辨率为亚皮秒（小于百万分之一秒）。其他实验，在单一蛋白质分子上进行，将确定光解酶如何结合和操纵受损的DNA链。许多复杂的工作将由研究生和研究生学者完成。其他重要的实验将由一批才华横溢的本科生进行。这些学生将获得广泛的经验，这是生物物理化学的标志。

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.