Most carcinogens and alkylating antineoplastic agents react with DNA to afford a diverse mixture of products. The complexity of the damage is a barrier to being able to quantitatively and qualitatively dissect out the biological role(s) of the individual DNA lesions relative to the mutagenic and toxic potency of the agent. To address this issue, we have prepared groove and sequence selective DNA methylating (Me-lex), ethylating (Et-lex) and 2-chloroethylating (CENU-lex) agents that in vitro and in vivo selectively afford minor groove 3-alkyladenine adducts. Our goal is to use this new class of minor groove alkylating agents to understand the role of 3-alkyladenine lesions in toxicity and mutagenicity.
The Specific Aims are to: (1) Determine using ligation-mediated PCR the in vivo alkylation patterns that are generated by Me-lex, Et-lex and CENU-lex in mouse ES cells that are wild type or null mutant for the glycosylase enzyme (Aag) that repairs 3-alkyladenine lesions; (2 and 3) Determine the relationship between DNA alkylation and apurinic sites, and the induction of mutations using a yeast shuttle vector assay. It is also proposed to evaluate the same endpoints in yeast strains that are deficient: (a) in the glycosylase that removes 3-alkyladenine from DNA; (b) in the AP endonuclease that processes abasic sites; and (c) in mismatch repair; (4) prepare DNA containing a single 3-alkyladenine substitution to determine the stability of 3- alkyladenine in DNA, if the lesions are substrates for nucleotide excision repair, and how DNA polymerization is effected by the presence of the adducts; (5) Determine why specific human cell lines are sensitive to Me-lex by assaying for their ability to repair DNA damage as measured by the time-dependent disappearance of 3-alkyladenine; and (6) Synthesize alkylating agents that will selectively generate the minor groove N3-methylguanine lesion and study their biological activity (toxicity and mutagenicity). In summary, we will be able to relate specific types of DNA damage to the site and type of mutation, and the enzymes that protect from the toxic and/or mutagenic response.
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