Mutagenic cytosine deamination is an intrinsic property of DNA; it occurs at a significant rate under physiological conditions. The repair of this DNA damage is usually initiated by uracil-DNA glycosylase, which excises the damaged base (uracil) from DNA. Cells lacking this enzyme have a high mutation rate. This enzyme presents an opportunity for understanding how enzymes specifically interact with DNA and how DNA repair enzymes function to prevent diseases, such as cancer, which appear to be a consequence of mutagenesis. The objectives of this research are to determine the protein structure, the substrate specificity and the nucleic acid binding properties of the E. coli DNA repair enzyme uracil-DNA glycosylase. 1) We will determine the DNA sequence (and thus the amino acid sequence) of uracil glycosylase and its surrounding transcriptional control sequences. 2-3) The unusual substrate specificity of uracil glycosylase is worth analyzing in detail to learn more about mechanisms by which enzymes dinstinguish between RNA and DNA. Uracil glycosylase must be able, simultaneously, to discriminate between uracil and thymine residues and to distinguish DNA from RNA. Specifically, we will use spectroscopic methods to determine the dissociation constants for potential substrates and inhibitors (normal RNA and DNA, uracil-containing DNA, and depyrimidinated-DNA) and test the detailed catalytic specificity for these and other substrates. 4) By determining the processiveness of uracil glycosylase we will learn how the enzyme monitors the genome for deaminated cytosine bases: by movement of the bound enzyme along the DNA or by repeated cycles of attachment to and dissociation from the DNA. 5) Mutant enzymes will be constructed by directed mutagenesis to test the importance of specific amino acid residues in catalytic and DNA binding properties of the enzyme.
| Duncan, B K (1985) Isolation of insertion, deletion, and nonsense mutations of the uracil-DNA glycosylase (ung) gene of Escherichia coli K-12. J Bacteriol 164:689-95 |
