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

Abstract

The sequence specificity of DNA damage of n-bromoalkylphenanthridinium bromides, with linker chain lengths ( n) of 4,6,8 and 10 methylene groups, was investigated in the plasmid pUC8 and in intact human cells. A linear amplification assay was used to elucidate the DNA sequence specificity of the alkylating agents. In this assay Taq DNA polymerase extends from an oligonucleotide primer up to the damage site and the products run on a DNA sequencing gel to reveal the precise sites of DNA damage. For both the plasmid and cellular experiments, the compound that caused the most damage to DNA was the n = 6 compound, followed by (in decreasing order) the n = 4, n = 8, and n = 10 compounds. There were significant differences in the sequence specificity of DNA damage between 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 damaged sequences were at 5′-Ga-3′ for the n = 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 human cells, the compounds produced damage with similar DNA sequence selectivity to that found in plasmid DNA. The n = 4 and 6 compounds possess marginal anti-tumour activity and these compounds produced the most damage in intact human cells. The n = 8 and 10 compounds do not demonstrate significant anti-tumour activity and these compounds resulted in the least damage in cells.