DNA Repair in Real Time: Ultrafast and Single Molecule Studies

Temple University, Philadelphia PA

Investigators

Abstract

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.