DNA sequence selectivity of guanine-N7 alkylation by nitrogen mustards is preserved in intact cells

摘要

Nitrogen mustard alkylating agents react with isolated DNA in a sequence selective manner, and the substituent attached to the drug reactive group can impose a distinct sequence preference. It is not ciear however to what extent the observed DNA sequence preferences are preserved in intact ceiis. The highly reiterated sequence of human alpha DNA has been used to determine the sites of guanlne-N7 aikylation following treatment of cells with three nitrogen mustards, mechiorethamine, uracil mustard and quinacnne mustard, known to react in isolated DNA with distinctly different sequence preferences. Alpha DNA from drug treated cells was extracted, purified, end-labeled, and a 296 base pair, singly end-labelled, fragment isolated. Following the quantitative conversion of alkyiation sites to strand breaks the fragments were separated on DNA sequencing gels. Clear differences were observed between the alkylatlon patterns of the three compounds, and the selectivitles were qualitatively similar to those predicted and observed in the same sequence alkylated in vitro. in particular the unique preferences of uracil and qulnacrine mustards for 5′-PyGC-3′ and 5′-T/GPu-3′ sequences, respectively, were preserved in intact cells suggesting that the pattern of sequence dependent reactivity Is not grossly affected by the nuclear milieu.