Title

In vitro and in vivo mutagenesis studies of site-specifically located DNA damages: Thymine glycol, urea, N-(guanin-8-yl)-1-aminopyrene, and ethenoadenine

Date of Completion

January 1999

Keywords

Biology, Genetics|Biology, Cell|Chemistry, Biochemistry

Degree

Ph.D.

Abstract

Chemical carcinogens, ionizing radiation, and endogenous metabolites produce a broad spectrum of damages to DNA. Some of these damages may trigger altered cellular function. Therefore, the cytotoxic and mutagenic potential of four DNA lesions, thymine glycol (t′), urea (u ′), N-(guanin-8-yl)-1-aminopyrene (dG AP), and ethenoadenine (ϵAde) are evaluated using a variety of biological assays. ^ A 26-mer template containing a single t′ or u ′ was constructed to study the in vitro DNA replication. Both t′ and u′ impeded DNA replication by several DNA polymerases. The specificity of nucleotide incorporation opposite the lesions and chain extension by 3′ → 5′ exonuclease-deficient Klenow fragment was determined by steady-state kinetic analysis. Kinetic studies revealed that the highest rate of incorporation for both the lesion sites was with dAMP. However, the efficiency of forming a G·T pair relative to a A·T pair for the control at a level of 1/109 was enhanced to approximately 1/160 for t′ and 1/20 for u′, although the former was more bypassable than the latter lesion. ^ Using recombinant DNA techniques, t′, u′ , dGAP, and ϵAde were incorporated into an M13 bacteriophage genome at a preselected site. The t′ or u′ modified single stranded M13 DNA were transfected into Escherichia coli and the cytotoxicity and mutagenicity were evaluated. Both t′ and u′ inhibited DNA synthesis in vivo. However, inhibitory effect of t ′ was less pronounced than the u′ lesion. We were unable to detect any mutations resulting from t′. In the case of u′, however, a variety of deletions were detected. Double stranded M13 DNA containing dGAP and ϵAde was replicated in a cell-free extract of human fibroblasts with the dGAP modified M13, a low frequency of deletions (0.2%) at the site of the lesion was observed. The in vitro replication results were consistent with the NMR and thermodynamic data that suggest stabilization of a −1 deletion intermediate. The mutation frequency for the site-specifically located ϵAde was 0.14%, with ϵAde → G being the main mutagenic event. The work presented in this thesis show that these four DNA lesions are mutagenic and/or cytotoxic. ^

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