Mechanisms and Models of DNA Photolyase

DNA photolyases are enzymes that repair the cyclobutane pyridine dimer (CPD) and 6-4 photoproduct lesions, formed through UV irradiation of DNA. These photolesions are the primary cause of skin cancer, which is with more than 2,000,000 patients per year in the US along a massive public health problem. DNA photolyases use a ETC mechanism that is unique in nature. Many aspects of the repair are under intense investigation in the scientific community, but are poorly understood.

Using different electronic structure methods as well as docking and MD techniques, my group investigated the physical basis for substrate recognition by CPD photolyase and developed a model of the enzyme-substrate complex that was widely recognized and is the current working hypothesis in the community.

Hybrid DFT calculations from my group, were also able to resolve several of the contradictions regarding the mechanism of the actual cycloreversion mechanism of the CPD radical anion. These calculations demonstrate that hydrogen bonding to the ketyl radical anion result in a valence state radical anion, which in turn undergoes essentially barrierless cycloreversion as shown on the right. Related experimental and computational work on the radical cationic cycloreversion mechanism of model systems validated the computational methodology and demonstrated the problems that need to be addressed in the interpretation of the experimental isotope effects

During the last two years, there has been significant progress in the design, synthesis, and evaluation of photolyase model systems. Both oxidative and reductive model systems were published and found widespread interest in the scientific community and the popular press. The oxidative photolyase model also has a very unusual crystal structure with a parallel arrangement of a highly polar molecule in a non-centrosymmetric crystal (shown on the right) and has therefore potentially interesting NLO properties. The reductive photolyase model was optimized to bind and repair CPD under physiological conditions (aqueous solution, pH7.2).

This last aspect has significant potential for further development through expansion towards biologically more relevant systems. We have started to study the CPD repair in the context of larger DNA segments and will follow up on initial results obtained by Beiersdorf that indicate activity in whole cells. The same recognition unit will also be used to develop a simple fluorometric assay for base flipping, which will in turn serve as a validation to ongoing large-scale MD simulations of base flipping in photodamaged DNA. Finally, future work in the area of DNA photolyase will include mechanistic studies of the 6-4 photolyase. We have obtained initial evidence that the published mechanism is highly unlikely and will pursue a combined computational and isotopic labeling study to support our proposed alternative mechanism.