DNA damage induced by bleomycin, neocarzinostatin, and melphalan in a precisely positioned nucleosome. Asymmetry in protection at the periphery of nucleosome-bound DNA.

The antitumor drugs bleomycin, neocarzinostatin, and melphalan all damage DNA by mechanisms which involve binding in the minor groove. In order to examine at high resolution the modulating effects of chromatin structure on the action of these drugs, an end-labeled DNA fragment from the Xenopus laevis 5 S rRNA gene was reconstituted with histone octamers to form a precisely positioned nucleosome. For each drug, DNA damage at specific sequence positions in the fragment was then compared for nucleosome-bound versus naked DNA. Reconstitution into nucleosomes resulted in a marked inhibition of the DNA cleavage induced by bleomycin (5-fold) and neocarzinostatin (2.4-fold) in the central region of nucleosomal DNA. However, at the periphery of nucleosome-bound DNA, a distinct asymmetry was apparent, with marked inhibition of cleavage toward the upstream side, but little if any inhibition toward the downstream side, which overlaps the binding site of the transcription factor TFIIIA. In the case of melphalan, alkylation at adenine N-3 was inhibited by nearly 2-fold throughout the nucleosome, whereas alkylation at guanine N-7 was either slightly inhibited or slightly enhanced, depending on sequence position. None of the drugs showed the 10-base pair periodicity characteristic of hydroxyl radical-induced cleavage of nucleosomal DNA. The results are consistent with a model in which minor groove sites in nucleosome-bound DNA remain relatively accessible to small molecules, even where the minor groove faces the histone core, and in which drug-induced DNA damage is inhibited by conformational constraints imposed on DNA by nucleosome structure. Furthermore, the degree of such constraints appears to be sequence-dependent, at least near the periphery of nucleosome-bound DNA.

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

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 thermolabile 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 mutagenesis. 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 (Hydropathic INTeractions) 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.

Thermolabile adenine adducts and A.T base pair substitutions induced by nitrogen mustard analogues in an SV40-based shuttle plasmid.

It was previously shown that the predominant mutations induced by melphalan (L-phenylalanine mustard) in the supF gene of shuttle plasmid pZ189 during replication in human cells are A.T----T.A transversions. In order to determine whether adenine adducts were formed at sequence positions corresponding to these mutations, melphalan-induced thermolabile adducts were mapped in the supF gene by selective depurination followed by strand cleavage in alkali. All A.T base pairs which were frequent sites for melphalan-induced A.T----T.A transversions were also prominent sites for formation of thermolabile adenine adducts. Although no mutations were detected at some prominent adduct sites, there was a significant correlation between adduct sites and mutation sites. While runs of two or more adenines were particularly prominent adduct sites, comparison of results obtained with 3'- and 5'-end-labeled DNA gave no evidence for intrastrand cross-links between adjacent adenines. Chlorambucil, another aromatic nitrogen mustard, showed sequence specificities for both mutagenesis and adenine adduct formation nearly identical to those seen with melphalan. The nonaromatic analogues mechlorethamine and phosphoramide mustard were much less efficient in inducing thermolabile adenine adducts, and mechlorethamine induced significantly fewer transversions at A.T base pairs than chlorambucil or melphalan. Formation of thermolabile adenine adducts by the aromatic nitrogen mustards was markedly reduced by blockage of the minor groove with distamycin, or by prior heat denaturation of the DNA. These results suggest that alkylation occurs primarily at the N-3 rather than N-7 position of adenine, probably as a consequence of the affinity of the aromatic rings of melphalan and chlorambucil for the minor groove.(ABSTRACT TRUNCATED AT 250 WORDS)