Dynamics and Mechanism of DNA-Repair Photolyase and Circadian Cryptochrome

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DESCRIPTION (provided by applicant): Photolyase is a photoenzyme that uses the energy of blue light to reverse UV-induced DNA damage in many organisms. Cryptochrome is a recently discovered blue-light photoreceptor that regulates the circadian clock in animals (and plants) and growth and development in plants. Both proteins have similar structural architectures but with totally different functions. We have recently elucidated the repair mechanisms and photocycles of cyclobutane pyrimidine dimer by a class of microbial photolyases (class-I) and of pyrimidine-pyrimidone (6-4) photoproduct by (6-4) photolyases. Several new classes of photolyases have been recently discovered with novel active sites and some new functions have also been lately observed. Thus, the first objective (Aim 1) of the project is to systematically characterize the repair of cyclobutane pyrimidine dimer by all other three classes of photolyases and the new function of Dewar repair by (6-4) photolyases. Such systematic investigations will obtain the molecular understanding of detrimental effects of UV radiation on the biosphere. Cryptochrome has been heavily studied by molecular genetics but the mechanistic investigation and understanding are simply lacking. Even the active (functional) redox state of cofactor flavin in cryptochrome is unknown yet. It is only known that cryptochrome proceeds to conformational changes to trigger downstream signal transduction upon blue-light photoreception. Thus, the second objective (Aim 2) is to systematically investigate the redox state(s) and related photochemistry, determine the active state in vivo in plant and animal cryptochromes, and characterize subsequent conformation dynamics and related interactions with downstream proteins. These investigations will uncover the primary process of initial signal transduction and reveal the reaction mechanism and photocycle of cryptochrome. To achieve these goals, we integrate state-of-the-art laser spectroscopy and biochemistry/molecular biology and follow the entire functional evolution of DNA repair and initial signaling of the complex processes with femtosecond temporal resolution and single-residue spatial resolution. The new knowledge obtained from this work on photolyase and cryptochrome is significant to the DNA repair and biological clock fields and, more importantly, is critical to practical applications of rug design for a series of diseases such as skin cancer and mental disorder.