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Cleavage of DNA with methidiumpropyl-EDTA-iron(II): reaction conditions and product analyses

Hertzberg, Robert P. and Dervan, Peter B. (1984) Cleavage of DNA with methidiumpropyl-EDTA-iron(II): reaction conditions and product analyses. Biochemistry, 23 (17). pp. 3934-3945. ISSN 0006-2960. https://resolver.caltech.edu/CaltechAUTHORS:20160511-153002453

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Abstract

The synthesis of methidiumpropyl-EDTA (MPE) is described. The binding affinities of MPE, MPE•Ni(II), and MPE•Mg(II) to calf thymus DNA are 2.4 X 10^4 M^(-1), 1.5 X 10^5 M^(-1), and 1.2 X 10^5 M^-1), respectively, in 50 mM NaCl, pH 7.4. The binding site size is two base pairs. MPE•Mg(II) unwinds PM2 DNA 11 ± 3° per bound molecule. MPE•Fe(II) in the presence of O_2 efficiently cleaves DNA and with low sequence specificity. Reducing agents significantly enhance the efficiency of the cleavage reaction in the order sodium ascorbate > dithiothreitol > NADPH. At concentrations of 0.1-0.01 µM in MPE•Fe(II) and 10 µM in DNA base pairs, optimum ascorbate and dithiothreitol concentrations for DNA cleavage are 1-5 mM. Efficient cleavage of DNA (10 µM in base pairs) with MPE•Fe(II) (0.1-0.01 µM) occurs over a pH range of 7-10 with the optimum at 7.4 (Tris-HC1 buffer). The optimum cleavage time is 3.5 h (22 °C). DNA cleavage is efficient in a Na^+ ion concentration range of 5 mM to 1 M, with the optimum at 5 mM NaCl. The number of single-strand scissions on supercoiled DNA per MPE•Fe(II) under optimum conditions is 1.4. Metals such as Co(II), Mg(II), Ni(II), and Zn(II) inhibit strand scission by MPE. The released products from DNA cleavage by MPE•Fe(II) are the four nucleotide bases. The DNA termini at the cleavage site are 5'-phosphate and roughly equal proportions of 3'-phosphate and 3'-(phosphogiycolic acid). The products are consistent with the oxidative degradation of the deoxyribose ring of the DNA backbone, most likely by hydroxy radical.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1021/bi00312a022DOIArticle
http://pubs.acs.org/doi/abs/10.1021/bi00312a022PublisherArticle
ORCID:
AuthorORCID
Dervan, Peter B.0000-0001-8852-7306
Additional Information:© 1984 American Chemical Society. Received November 29, 1983. This is Contribution No. 6953 from the Laboratories of Chemistry. This work was supported by Grant GM-27681 from the National Institutes of Health and a National Research Service Award (GM-07616) to R.P.H.
Funders:
Funding AgencyGrant Number
NIHGM-27681
NIH Predoctoral FellowshipGM-07616
Issue or Number:17
Record Number:CaltechAUTHORS:20160511-153002453
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20160511-153002453
Official Citation:Cleavage of DNA with methidiumpropyl-EDTA-iron(II): reaction conditions and product analyses Robert P. Hertzberg and Peter B. Dervan Biochemistry 1984 23 (17), 3934-3945 DOI: 10.1021/bi00312a022
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:67026
Collection:CaltechAUTHORS
Deposited By: Victoria Brennan
Deposited On:19 May 2016 22:00
Last Modified:26 Nov 2019 11:15

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