The occurrence of uracil residues in DNA is an important human heatth concern because increased levels of uracil-DNA can lead to mutagenesis and malignant transformation. Uracil may also play a role in the deleterious accumulation of mitochondrial mutations associated with human degenerative diseases such as Parkinson's disease and diabetes, and with human aging. The long term objective of this research proposal is centered on understanding the impact of uracil-DNA accumulation on human health and disease. Our knowledge of the frequency of dUMP incorporation into mitochondrial and nuclear DNA is quite limited. In addition, little is known concerning the rate of cytosine deamination in the mitochondrial oxidative environment as well as in chromatin, nor has the effect of enviromental agents on this premutagenic process been adequately characterized. This proposal focuses on four specific aims designed to elucidate the dynamics of uracil accumulation in human cellular DNA. First, the extent to which uracil residues (U'A and U'G) accumulate in mitochondrial and nuclear DNA will be determined using a recently developed sensitive method for detecting uracil sites in DNA. Second, the concentration of premutagenic U?G lesions produced by cytosine deamination in mitochondrial and nuclear DNA will be determined using E. coli doublestrand specific uracil-DNA glycosylase (UNG). The question whether uracil is distributed randomly about the mitochondrial genome or occurs in specific regions of the DNA, such as the origin of mitochondrial replication, will be investigated. Third, the significance of uracil-DNA glycosylase (UNG) instigated base excision repair in uracil avoidance will be assessed by examining uracil-DNA accumulation in the mitochondrial and nuclear genomes of UNG-defective human glioblastoma cells. Fourth, the contribution of alternate uracil-excision activities such as TDG, SMUG1, and MBD4, to the uracil-initiated base excision DNA repair pathway will be investigated in extracts of human cells. The results of this proposed research will shed light on mitochondrial and nuclear uracil-DNA metabolism and provide the foundation for future studies aimed at elucidating the role of genome-specific uracil-DNA repair in mutation avoidance.
