McHugh M M, Woynarowski J M, Mitchell M A, Gawron L S, Weiland K L, Beerman T A
Department of Experimental Therapeutics, Roswell Park Cancer Institute, Buffalo, New York 14263.
Biochemistry. 1994 Aug 9;33(31):9158-68. doi: 10.1021/bi00197a019.
CC-1065 is a minor-groove bonding agent capable of forming covalent adducts with the N-3 position of adenines within A-T-rich regions of duplex DNA. By examining the formation and location of CC-1065 adducts within the simian virus 40 (SV40) DNA molecule, the present study marks the first time that the precise sites of CC-1065 lesions have been identified at the level of eukaryotic genomic DNA. In naked DNA preparations, r values (moles of drug/mole of nucleotide base pair) > or = 0.0015 effected, after thermal treatment, a measurable decrease in intact supercoiled form I, as well as increases in forms II and III, indicating that both single-strand and apparent double-strand damage had occurred. A similar pattern of damage was observed in SV40-infected cells, albeit at higher CC-1065 levels. The amount of CC-1065 required to produce a 50% loss in form I was > 2-fold higher in infected cells (r = 0.029) than with purified DNA samples (r = 0.013). The appearance of double-strand damage at low drug levels suggested a high specificity of CC-1065 bonding to localized regions of the genome. The precise location of these CC-1065 adduction sites was examined by three methods: sequence analysis of the entire genome (GenBank), DNA polymerase termination assay of specific fragments of SV40, and restriction enzyme digestion analysis of the entire SV40 molecule. When sequence analysis of the entire genome was performed by examining both strands for the presence of the consensus CC-1065 binding sequence 5'-A/T-A/T-A/T-A/T-A*-3'[Reynolds et al. (1985) Biochemistry 24, 6228-6247], 294 single-strand adduction sites were predicted, compared to 20 sites where CC-1065 should bond to both strands within a 30-base-pair window and at which, when heated, a double-strand break should occur. DNA polymerase termination assay of actual adduction sites was performed on restriction fragments of SV40 DNA pretreated with CC-1065 in infected cells or in purified supercoiled DNA preparations and selected on the basis of the sequence analysis (i.e., regions 2510-2730, 3701-3920, 4400-4659, 4020-4320, and 5163-65). In general, double-strand lesions were detected in similar regions of the genome by the DNA termination assay and by sequence analysis. When restriction enzyme digestion and the DNA polymerase termination assay were compared throughout the genome, nearly identical patterns of adduct formation were observed. Interestingly, similar alkylation patterns were observed with either naked or infected cell DNA.(ABSTRACT TRUNCATED AT 400 WORDS)
