PubMed · 2223396
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T A Connors. 1990. Alkylating agents.. https://pubmed.ncbi.nlm.nih.gov/2223396/
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This report presents evidence that a reduced pyrrolo[1,2-a]benzimidazole (PBI) cleaves DNA as a result of phosphate alkylation followed by hydrolysis of the resulting phosphate triester. The base-pair specificity of the phosphate alkylation results from Hoogsteen-type hydrogen bonding of the reduced PBI in the major groove at only A.T and G.C base pairs. Alkylated phosphates were detected by 31P NMR and the cleavage products were detected by 1H NMR and HPLC. Evidence is also presented that a reduced PBI interacts with DNA in the major groove rather than in the minor groove or by intercalation.
Intermolecular triplex DNA is stabilized by metal cations and polyamines which reduce repulsion between the negatively charged phosphates of the three nucleic acid strands. We use a quantitative chemical-probing assay involving protection of duplex guanines in a homopyrimidine.homopurine (Py.Pu) sequence from dimethyl sulfate modification to study effects of basic oligopeptides on the stability of triplex DNA. An intermolecular protonated pyrimidine.purine.pyrimidine (Py.Pu*Py) triplex formed readily between a duplex DNA region and a 14-mer pyrimidine triplex-forming oligonucleotide (TFO) at pH 5. The triplex was stabilized at pH by the addition of magnesium ions. In the presence of spermine and lysine-rich peptides, the intermolecular triplex was stabilized up to pH 6.5-7.0. The effective peptide concentration required for stabilization was 10(-5)-10(-2) M. Of the basic peptides studied, pentalysine (Lys-Lys-Lys-Lys-Lys) was the most effective triplex stabilizer. It was effective at concentrations which are lower than those required for Lys-Gly-Lys-Gly-Lys and Lys-Ala-Lys-Ala-Lys and are similar to active concentrations of spermine. Basic peptides were more effective at stabilizing a Py.Pu*Py triplex than a pyrimidine.purine.purine (Py.Pu*Pu) triplex. At 1 mM, Lys-Lys-Lys-Lys-Lys stabilized the Py.Pu*Pu triplex at a level comparable to stabilization by Mn2+ and spermine, whereas Lys-Gly-Lys-Gly-Lys and Lys-Ala-Lys-Ala-Lys resulted in weaker TFO binding. The concentration of TFOs required to form triplex DNA were significantly reduced in the presence of peptides.(ABSTRACT TRUNCATED AT 250 WORDS)
The bifunctional sulphur mustard (bis-(2-chloroethyl)sulphide, HD) and its monofunctional analogue (2-chloroethyl ethyl sulphide, CEES) are both vesicants. In this study, both mustards were shown to rapidly alkylate the AP2 consensus binding sequence incorporated in a 26mer oligonucleotide. The reaction was essentially complete within 10 min under the conditions employed in this study and -95% of the oligonucleotides were alkylated at least once using 500 microM HD and 1 mM CEES. Progressive alkylation of the consensus sequence was parallelled by a decrease in transcription factor binding. Under reaction conditions which alkylated approximately 95% of the oligonucleotides at least once, the binding of cloned human AP2 was reduced by 93 and 76% by HD and CEES, respectively, compared with control values. The interference with binding is a result of alkylation of the DNA and not damage to the transcription factor by mustard or its hydrolysis products. Interference with transcription factor binding would be expected to have a profound influence on the ability of the cell to function normally and to respond to DNA damage and may contribute significantly to the skin damage produced by these compounds.