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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM066245-01
Application #
6533262
Study Section
Physiological Chemistry Study Section (PC)
Program Officer
Wolfe, Paul B
Project Start
2002-08-01
Project End
2007-07-31
Budget Start
2002-08-01
Budget End
2003-07-31
Support Year
1
Fiscal Year
2002
Total Cost
$247,987
Indirect Cost
Name
Oregon State University
Department
Public Health & Prev Medicine
Type
Schools of Earth Sciences/Natur
DUNS #
053599908
City
Corvallis
State
OR
Country
United States
Zip Code
97339
Bennett, Samuel E; Kitner, Joshua (2006) Characterization of the aldehyde reactive probe reaction with AP-sites in DNA: influence of AP-lyase on adduct stability. Nucleosides Nucleotides Nucleic Acids 25:823-42
Lari, Sibghat-Ullah; Chen, Cheng-Yao; Vertessy, Beata G et al. (2006) Quantitative determination of uracil residues in Escherichia coli DNA: Contribution of ung, dug, and dut genes to uracil avoidance. DNA Repair (Amst) 5:1407-20
Chen, Cheng-Yao; Mosbaugh, Dale W; Bennett, Samuel E (2005) Mutations at Arginine 276 transform human uracil-DNA glycosylase into a single-stranded DNA-specific uracil-DNA glycosylase. DNA Repair (Amst) 4:793-805
Ko, Rinkei; Bennett, Samuel E (2005) Physical and functional interaction of human nuclear uracil-DNA glycosylase with proliferating cell nuclear antigen. DNA Repair (Amst) 4:1421-31
Bennett, Samuel E; Chen, Cheng-Yao; Mosbaugh, Dale W (2004) Escherichia coli nucleoside diphosphate kinase does not act as a uracil-processing DNA repair nuclease. Proc Natl Acad Sci U S A 101:6391-6
Chen, Cheng-Yao; Mosbaugh, Dale W; Bennett, Samuel E (2004) Mutational analysis of arginine 276 in the leucine-loop of human uracil-DNA glycosylase. J Biol Chem 279:48177-88