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. ^