The major objectives of this proposal are to determine the importance of specific types of DNA damage for cell lethality and mutagenicity and to study the repair of these DNA adducts in cells. In order to understand the biological roles of the DNA adducts and to investigate the particular gene involved in their repair, Phix174 am3 RF DNA will be modified in vitro with chemical mutagens or carcinogens to produce specific types of DNA addusts. These DNA will be used to transfect E.coli cells with mutation in uvrA, uvrB, uvrC or alk gene to survey the lethality and mutagenicity of these adducts. We have found that 1) uvr genes function differently in the repair of 2-acetylaminofluorene (AAF) and 2-aminofluorene (AF) DNA adducts and 2) AF and 9r, 10t-dihydroxy-7c,8c-oxy-7,8,9,10-tetrahydrobenzo(a)pyrene (BPDE-III) DNA adducts are much less lethal than AAF, two other BPDE steric isomer DNA adducts or pyrimidine dimers. We will explore the biochemical basis for these differences by examining a) how uvrA, B, C, and alk gene products react with these adducts and b) the effect of these DNA adducts in DNA replication. The second major study is designed to investigate the repair of chemical agent induced DNA damage in mammalian cells, specifically in the thymidine kinase gene among five classes of Chinese hamster ovary (CHO) UV sensitive mutants using DNA mediated gene transfer techniques. We have found that (deoxyquanosin-c8-yl)-2-aminofluorene (dG-C8-AF) is the major DNA adduct formed in human and CHO cells treated with N-acetoxy-2-acetylaminofluorene (NA-AAF). Since in E. coli cells, the uvrC gene is responsible for the repair of dG-C8-AF, the NA-AAF sensitive xeroderma pigmentsum (XP) group A, C, D, & E cells and Class 1 & 11 CHO UV sensitive mutants may be defective in a uvrC type of gene function. We will explore this possibility by examining whether a) uvrC protein (singly or in combination with uvrA and uvrB proteins) will complement the repair of chemical and UV damage in permeable XP cells and B) whether transfected prokaryotic/eukaryotic PSV2qpt-uvrC vector can complement the deficiency of xp and UV sensitive CHO cells. We will examine the rate and extent of removal of the imidazole ring-opened dG-C8-AF adduct adducts among five classes of CHO mutant cells. The result of all these studies should provide a better understanding of the strategies human cells use to repair chemical damage to DNA.
|Denissenko, M F; Chen, J X; Tang, M S et al. (1997) Cytosine methylation determines hot spots of DNA damage in the human P53 gene. Proc Natl Acad Sci U S A 94:3893-8|