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)
Wang
P
P
Department of Pharmacology and Toxicology, Medical College of Virginia, Richmond 23298-0230.
Bauer
G B
GB
Bennett
R A
RA
Povirk
L F
LF
D000227:Adenine Nucleotides / Q000235:genetics*; D000477:Alkylating Agents / Q000494:pharmacology; D001482:Base Composition; D001483:Base Sequence; D002699:Chlorambucil / Q000494:pharmacology; D005822:Genetic Vectors; D006358:Hot Temperature; D008466:Mechlorethamine / Q000031:analogs & derivatives / Q000494:pharmacology*; D008558:Melphalan / Q000494:pharmacology; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010957:Plasmids / Q000187:drug effects; D013539:Simian virus 40 / Q000235:genetics*; D013942:Thymine Nucleotides / Q000235:genetics*
D000227:Adenine Nucleotides; D000477:Alkylating Agents; D013942:Thymine Nucleotides; D002699:Chlorambucil; D008466:Mechlorethamine; D008558:Melphalan