DNA adenine adducts induced by nitrogen mustards and their role in transcription termination in vitro

Abstract

Previous studies have demonstrated that three cancer chemotherapeutic compounds of the nitrogen mustard class, melphalan (L-PAM), nitrogen mustard (HN2) and chlorambucil (CBC), each generated DNA lesions that prematurely terminate in vitro transcription. Sites of these lesions were inconsistent with sites of N7 guanine monoadducts formed by these compounds, and in the cases of L-PAM and CBC were suggestive of adenine lesions. The present study is an attempt to identify and characterize nitrogen mustard-induced non-N7 guanine DNA adducts, and in particular adenine DNA adducts, and to assess their role in drug-induced in vitro transcription termination. Data from studies using a modified Maxam-Gilbert DNA sequencing technique demonstrate that L-PAM and CBC, but not HN2, generate heat-labile, alkaline-stabilized adenine adducts at nearly every adenine in a region of a defined DNA template examined. Comparison of sites of L-PAM- and CBC-induced adenine adducts to known sites of drug-induced transcription termination in the same DNA template show that L-PAM- and CBC-induced transcription termination is associated not with drug lesions at single adenines, but rather with drug-induced adducts at neighboring adenines. Additional in vitro transcription studies using a small DNA molecule generated by polymerase chain reaction-mediated DNA amplification demonstrate that none of the transcription-terminating lesions induced by L-PAM and CBC in this molecule are interstrand in nature. These results suggest that some, but not all, nitrogen mustard compounds can generate heat-labile adenine lesions in DNA, and that bifunctional nitrogen mustards that can form heat-labile adenine adducts also form adducts consistent with intrastrand adenine-adenine crosslinks. These adducts at pairs of adenines in turn appear to be responsible for L-PAM and CBC-induced transcription termination in vitro.