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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.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:Chemistry
Degree Grantor:California Institute of Technology
Division:Chemistry and Chemical Engineering
Major Option:Chemistry
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Grubbs, Robert H.
Thesis Committee:
  • Dervan, Peter B. (chair)
  • Gray, Harry B.
  • Richards, John H.
  • Grubbs, Robert H.
Defense Date:7 November 1983
Record Number:CaltechTHESIS:10102018-114702204
Persistent URL:https://resolver.caltech.edu/CaltechTHESIS:10102018-114702204
DOI:10.7907/vcna-tp03
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:11225
Collection:CaltechTHESIS
Deposited By: Lisa Fischelis
Deposited On:12 Oct 2018 17:08
Last Modified:19 Apr 2021 22:37

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