Effect of distamycin on chlorambucil-induced mutagenesis in pZ189: evidence of a role for minor groove alkylation at adenine N-3

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Abstract
Previous work has shown that the bifunctional alkylating agent chlorambucil induces thermolabile adenine adducts and that the predominant chlorambucil-induced mutations in shuttle vector pZ189 are transversions at AT base pairs. In order to assess the role of thermolabile adducts in generating these transversions, pZ189 was treated with chlorambucil in the presence of distamycin, which specifically blocks formation of themolabile adenine adducts. Analysis of the mutations resulting from replication of the damaged vector in human 293 cells showed that base substitutions at AT base pairs were specifically suppressed in concert with suppression of thermolabile adducts at specific sites in the supF target gene, strongly supporting a role for these adducts in muta-genesis. Since there is considerable evidence that these adducts are N-3 alkylations, a computer graphics model of such an adduct was constructed. Modeling studies indicated that the adduct could be formed with little distortion of the DNA helix. Analysis of the adduct using the HINT (Hydrophathic INTer-actions) program was consistent with the proposal that favorable hydrophobic interactions of the phenyl ring of chlorambucil with the wall of the minor groove may promote adenine N-3 alkylation by this drug.

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