THE INFLUENCE OF LINKER CHAIN-LENGTH ON THE SEQUENCE SPECIFICITY OF DNA-DAMAGE BY N-BROMOALKYLPHENANTHRIDINIUM BROMIDES IN PLASMID DNA AND IN INTACT HUMAN-CELLS

The sequence specificity of DNA damage of n-bromoalkylphenanthridinium bromides, with linker chain lengths (n) of 4,6,8 and 10 methylene gro ups, was investigated in the plasmid pUC8 and in intact human cells. A linear amplification assay was used to elucidate the DNA sequence spe cificity of the alkylating agents. In this assay Taq DNA polymerase ex tends from an oligonucleotide primer up to the damage site and the pro ducts run on a DNA sequencing gel to reveal the precise sites of DNA d amage. For both the plasmid and cellular experiments, the compound tha t caused the most damage to DNA was the n = 6 compound, followed by (i n decreasing order) the pi = 4, n = 8, and n = 10 compounds. There wer e significant differences in the sequence specificity of DNA damage be tween n-bromoalkylphenanthridinium bromides of different linker chain length: (1) the main sites of damage were at guanines for the n = 4,6 and 8 compounds but at guanines and adenines for the n = 10 compound; (2) a consensus sequence of 5'-c(a/t)Ggg-3' was obtained for the n = 4 ,6 and 8 compounds but 5'-c(a/c)(G/A)(g/a)-3' for the n = 10 compound; (3) runs of consecutive Gs were the major site of damage for the n = 4,6 and 8 compounds, but consecutive Gs or consecutive As for the n = 10 compound; (4) for damage at single isolated guanines, the most dama ged sequences were at 5'-Ga-3' for the it = 4 compound but at 5'-Gt-3' for the n = 6,8 and 10 compounds, The tandemly repeated alpha RI DNA sequence was the DNA target in intact human K562 cells. In intact huma n cells, the compounds produced damage with similar DNA sequence selec tivity to that found in plasmid DNA. The n = 4 and 6 compounds possess marginal anti-tumour activity and these compounds produced the most d amage in intact human cells. The n = 8 and 10 compounds do not demonst rate significant anti-tumour activity and these compounds resulted in the least damage in cells.