SPECIFICITY AND KINETICS OF INTERSTRAND AND INTRASTRAND BIFUNCTIONAL ALKYLATION BY NITROGEN MUSTARDS AT A G-G-C SEQUENCE

Previous work showed that melphalan-induced mutations in the aprt gene of CHO cells are primarily transversions and occur preferentially at G-G-C sequences, which are potential sites for various bifunctional al kylations involving guanine N-7. To identify the DNA lesion(s) which m ay be responsible for these mutations, an end-labeled DNA duplex conta ining a frequent site of melphalan-induced mutation in the aprt gene w as treated with melphalan, mechlorethamine or phosphoramide mustard, T he sequence specificity and kinetics of formation of bath interstrand and intrastrand crosslinks were determined, Ail mustards selectively f ormed two base-staggered interstrand crosslinks between the 5' G and t he G opposite C in the 5' G-G-C sequence, Secondary alkylation was muc h slower for melphalan than for the other mustards and the resulting c rosslink was more stable, Mechlorethamine and phosphoramide mustard in duced intrastrand crosslinks between the two contiguous Gs in the G-G- C sequence in double-stranded DNA, but melphalan did not, Molecular dy namic simulations provided a structural explanation for this differenc e, in that the monofunctionally bound intermediates of mechlorethamine and phosphoramide mustard assumed thermodynamically stable conformati ons with the second arm in a position appropriate for intrastrand cros slink formation, while the corresponding melphalan monoadduct did not.