Reactive oxygen species (ROS) produced from cellular metabolism and exposure to environmental agents pose a significant threat to genomic stability. Direct DNA oxidation by ROS or modification by products of lipid peroxidation result in the formation of mutagenic DNA adducts that are implicated in carcinogenesis. A variety of DNA adducts are produced following DNA oxidation including 3-(2!-deoxy- "-D-erythro-pentofuranosyl)- pyrimido[1,2-a]purin-10(3H)-one (M1dG) and N6-oxopropenyl-dA (OPdA). M1dG and OPdA possess the unique characteristic of electrophilic reactivity, which enables them to react with a variety of nucleophiles. OPdA is the more reactive adduct and forms covalent cross-links with N#-acetyl lysine. This raises the possibility that OPdA forms cross-links with DNA-binding proteins. The chemistry of this DNA-protein cross-link formation is not well understood and presents an exciting and novel area for discovery. I propose to use EcoRI as a model DNA-binding protein to elucidate the chemistry of cross-link formation and to optimize the methods for detection and identification of adducted proteins. I will then identify the proteins cross-linked to OPdA adducts in nuclear extracts of mammalian cells. The formation of covalent cross-links between DNA damage and protein has not been extensively investigated despite the detrimental cellular effects these conjugates can induce. The studies outlined in this proposal will increase our understanding of how cross-links are formed as well as what proteins are able to form cross-links with OPdA. This investigation represents the first study to elucidate the interaction of OPdA with DNA-binding proteins.

Public Health Relevance

The production of reactive oxygen species occurs during normal cellular functions and is increased by chronic inflammation and exposure to environmental agents. DNA is a target for attack by reactive oxygen and the damage produced has been linked to carcinogenesis. These studies will expand our understanding of how DNA damage induced by reactive oxygen contributes to carcinogenesis by examining endogenous DNA lesions that exhibit the ability to form cross-links to DNA-binding proteins.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Postdoctoral Individual National Research Service Award (F32)
Project #
5F32CA159701-02
Application #
8316447
Study Section
Special Emphasis Panel (ZRG1-F09-E (20))
Program Officer
Jakowlew, Sonia B
Project Start
2011-09-01
Project End
2014-08-31
Budget Start
2012-09-01
Budget End
2013-08-31
Support Year
2
Fiscal Year
2012
Total Cost
$52,190
Indirect Cost
Name
Vanderbilt University Medical Center
Department
Biochemistry
Type
Schools of Medicine
DUNS #
004413456
City
Nashville
State
TN
Country
United States
Zip Code
37212
Shuck, Sarah C; Wauchope, Orrette R; Rose, Kristie L et al. (2014) Protein modification by adenine propenal. Chem Res Toxicol 27:1732-42
Shuck, Sarah C; Rose, Kristie L; Marnett, Lawrence J (2014) Mass spectrometric methods for the analysis of nucleoside-protein cross-links: application to oxopropenyl-deoxyadenosine. Chem Res Toxicol 27:136-46