Ultraviolet light in sunlight crosslinks pyrimidines in cellular deoxyribonucleic acid (DNA), producing pyrimidine dimers. These lesions in DNA are thought to be a potential cause of solar-UV-induced skin cancer. Repair of the damage is carried out by remarkable enzymes that utilize visible light to split the dimers, thereby regenerating the original pyrimidines. The mechanism of this novel photobiological repair system is unknown, but it appears to involve a photoinitiated electron transfer from an enzyme-bound cofactor to enzyme-bound dimer. The electron transfer generates oppositely charged species and results in the splitting of the dimer, which constitutes repair of the DNA. It now appears that the enzymes may achieve their high efficiency of repair of DNA by preventing opposite charges from recombining in the active site of the enzyme. There are several ways in which they might accomplish this, such as optimization of active site polarity, insertion of physical barriers or distance between charges, electron transfer energetics, migration of charges, neutralization of one of the charges, and adduct formation. Which, if any, of these possibilities is employed by the repair enzymes is unknown.
The specific aims of this proposal are the study of each of these possible modes of interference with charge recombination in carefully designed systems that """"""""isolate"""""""" each mode for evaluation. The methodology includes the use of steady-state photolysis methods for evaluation of efficiency of the charge-recombination-blocking modes described above and laser flash photolysis methods for study of transient intermediates. The results of these studies will assist in formulation of an overall picture of how the enzymes work, what problems they face in carrying out their specific catalytic reaction, and how evolution shaped their mode of action. An additional specific aim of this work is to evaluate the biological effects of dimers in cells. This will be accomplished by use of artificial agents that are designed to repair dimers in cells that have been exposed to ultraviolet light. By comparing cells in which dimers have been repaired to cells in which they have not, the specific biological effects of pyrimidine dimers will be identified. This will ultimately lead to a better understanding of the potential role of dimers as causes of cancer.
