Functional Dynamics and Molecular Mechanism of Photolayse

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DESCRIPTION (provided by applicant): Functional Dynamics and Molecular Mechanism of Photolyase Photolyase is a photoenzyme that uses the energy of blue light to reverse UV-induced DMA damage in many organisms. It is lacking in humans, but recent experiments in human cell lines and in mice indicate that through gene therapy, photolyase can be used to heighten resistence to sunlight-induced tumorigenesis. Cyclobutane pyrimidine dimer (CPD) and pyrimidine-pyrimidone (6-4) photoproduct are the two major classes of cytotoxic, mutagenic and carcinogenic DNA photoproducts produced by UV-light irradiation of cells and are the main cause of skin cancer. A hypothetical molecular model of DNA repair by photolyase has been proposed and investigated for a long time, but direct evidence for the catalytic pathway had never been observed until last year when we captured a catalytic intemediate using ultrafast spectroscopy. Understanding of the remarkably efficient repair mechanism at the atomic scale has been lacking. Thus, the first objective of the project is to reveal the molecular mechanism of dimer repair in DNA by CPD photolyase at the most fundamental level and determine the key factors for its high repair efficiency. The mechanism of the (6-4)-photoproduct repair by (6-4) photolyase has been less examined and the repair dynamics, less efficient than CPD photolyase, have never been explored. Therefore, the second objective is to characterize the repair dynamics of (6-4) photolyase, elucidate its molecular mechanism, and understand the factors contributing to its lessened efficiency compared to CPD photolyase. To achieve these goals, we integrate state-of-the-art femtosecond laser spectroscopy, molecular biology methods and computational studies, and break down the dynamic complexity of repair processes into elementary steps. With femtosecond temporal resolution and single-residue spatial resolution, we will unravel the complete evolution of the functional dynamics and lay bare the fundamental mechanisms of DNA repair by photolyase. The understanding gained from this work will help in rational drug design and gene therapy to prevent skin cancer induced by UV light.