Cleavage of DNA with Methidiumpropyl-EDTA

Citation

Hertzberg, Robert P. (1984) Cleavage of DNA with Methidiumpropyl-EDTA. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/vcna-tp03. https://resolver.caltech.edu/CaltechTHESIS:10102018-114702204

Abstract

Attachment of ethylenediaminetetraacetate (EDTA) to the DNA intercalator methidium creates an efficient DNA cleaving molecule, methidiumpropyl-EDTA (MPE). MPE·Fe(II) (10^-7 M) single strand cleaves supercoiled pBR-322 plasmid DNA (10^-5 M) in the presence of O₂, converting it to 56% open circular DNA. In the presence of 1 mM dithiothreitol (DTT) and O₂, MPE·Fe(II) (10^-8 M) converts supercoiled pBR-322 DNA (10^-5 M) to 97% open circular and 3% linear DNA. MPE·Mg(II) binds to sonicated calf thymus DNA with a binding affinity of 1.2 x 10^-5 M^-1 and binding site size of 1.9 base pairs, and unwinds supercoiled PM2 plasmid DNA with an unwinding angle of 11° ± 3°.

The reaction conditions for DNA cleavage and factors affecting the cleavage efficiency by MPE·Fe(II) have been determined. The cleavage is dependent on Fe(II) and O₂, inhibited by chelating agents, enhanced by reducing agents (ascorbate > DTT > NADH), inhibited by catalase, partially inhibited by radical scavengers, relatively unaffected by sodium concentration, and optimum at pH 7.4 (in Tris·HCl buffer). MPE·Fe(II) cleaves DNA in a relatively non-sequence specific manner, with significantly lower sequence specificity than the enzyme DNAse I, and is a useful footprinting tool for the determination of small molecule binding sites on naturally occurring heterogeneous DNA.

The products from the cleavage reaction of MPE·Fe(II) with DNA have been characterized. The results demonstrate that each strand scission produces a free nucleotide base, a 5' phosphoryl group, and a mixture of 3' phosphoryl and 3' phosphoglycolic acid groups left on the polynucleotide chain. Very little malondialdehyde or base-propenals are produced. These products are consistent with the intermediacy of hydroxyl radical in the strand scission reaction.

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