CC-1065 BONDING TO INTRACELLULAR AND PURIFIED SV40 DNA - SITE-SPECIFICITY AND FUNCTIONAL-EFFECTS

CC-1065 is a minor-groove bonding agent capable of forming covalent ad ducts with the N-3 position of adenines within A-T-rich regions of dup lex DNA. By examining the formation and location of CC-1065 adducts wi thin the simian virus 40 (SV40) DNA molecule, the present study marks the first time that the precise sites of CC-1065 lesions have been ide ntified at the level of eukaryotic genomic DNA. In naked DNA preparati ons, r values (moles of drug/mole of nucleotide base pair) greater tha n or equal to 0.0015 effected, after thermal treatment, a measurable d ecrease in intact supercoiled form I, as well as increases in forms II and III, indicating that both single-strand and apparent double-stran d damage had occurred. A similar pattern of damage was observed in SV4 0-infected cells, albeit at higher CC-1065 levels. The amount of CC-10 65 required to produce a 50% loss in form I was >2-fold higher in infe cted cells (r = 0.029) than with purified DNA samples (r = 0.013). The appearance of double-strand damage at low drug levels suggested a hig h specificity of CC-1065 bonding to localized regions of the genome. T he precise location of these CC-1065 adduction sites was examined by t hree methods: sequence analysis of the entire genome (GenBank), DNA po lymerase termination assay of specific fragments of SV40, and restrict ion enzyme digestion analysis of the entire SV40 molecule. When sequen ce analysis of the entire genome was performed by examining both stran ds 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 windo w and at which, when heated, a double-strand break should occur. DNA p olymerase termination assay of actual adduction sites was performed on restriction fragments of SV40 DNA pretreated with CC-1065 in infected eels or in purified supercoiled DNA preparations and selected on the basis of the sequence analysis (i.e., regions 2510-2730, 3701-3920, 44 00-4659, 4020-4320, and 5163-65). In general, double-strand lesions we re detected in similar regions of the genome by the DNA termination as say and by sequence analysis. When restriction enzyme digestion and th e DNA polymerase termination assay were compared throughout the genome , nearly identical patterns of adduct formation were observed. Interes tingly, similar alkylation patterns were observed with either naked or infected cell DNA. The sites predicted by sequence analysis and obser ved in infected cells or with purified supercoiled SV40 DNA were local ized to specific functional regions of the genome associated with atta chment of SV40 DNA to the matrix and with DNA replication and separati on of daughter molecules, as well as with production of essential vira l proteins. Functional effects of CC-1065 adduct formation in SV40 inc luded a decrease in SV40 DNA accumulation. When cells were drug treate d at 2 h postinfection and then further incubated until 40 h postinfec tion, 50% and 100% decreases in accumulation of SV40 DNA were observed at 2.5 and 10 nM CC-1065, respectively. The formation of nascent SV40 DNA intermediates also was reduced, but not completely eliminated, in the presence of the drug. Each of the intermediates was inhibited to a similar extent, suggesting that drug action affected an early stage of replication.