The long term goals of the project are to utilize chemical and mechanistic approaches to understand biological systems that have a direct impacct on human health and disease. DNA carries life's genetic information encoded in the arrangement of bases along the length of the DNA molecule. Unfortunately, these bases are also chemically reactive and therefore susceptible to modification. Spontaneous change, damage by endogenous metabolites, and exposure to environmental agents such as carcinogens can alter the primary structure of DNA. The processes that maintain genomic integrity are vitally important for the fidelity of life. Base excision repair initiated by DNA glycosylases helps to maintain genomic stability by removing damaged bases from the genome. In order to perform their function, the DNA glycosylases must locate damaged base substrates amongst vast tracts of undamaged DNA.
The specific aims of the project are to determine the mechanism by which DNA glycosylases locate substrates in DNA. The mechanism might be either processive or distributive. A processive mechanism entails the diffusion of the enzyme along the DNA, maintiaining contact and scanning for substrates. A distributive mechanism involves the association and dissociation of the enzyme as it diffuses in three dimensions. The plan is to utilize substrate-containing DNA that is designed to test distributive or processive models for substrate recognition. Glycosylase activity will be followed using gel-based assays, and circular dichroism will be used to detect conformational changes in the glycosylase or the DNA. When the mechanism is successfully determined, the biological relevance of the model will be tested by studying specific mutants of DNA glycosylases in a model organism such as E. coli. Understanding precise mechanisms will lead to understanding which mutations in DNA repair genes cause alterations in function, and thus, lead to a decrease in genomic stability.
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