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

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

R P Hertzberg et al. Biochemistry. 1984.

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 degrees per bound molecule. MPE.Fe(II) in the presence of O2 efficiently cleaves DNA and with low sequence specificity. Reducing agents significantly enhance the efficiency of the cleavage reaction in the order sodium ascorbate greater than dithiothreitol greater than NADPH. At concentrations of 0.1-0.01 microM in MPE.Fe(II) and 10 microM in DNA base pairs, optimum ascorbate and dithiothreitol concentrations for DNA cleavage are 1-5 mM. Efficient cleavage of DNA (10 microM in base pairs) with MPE.Fe(II) (0.1-0.01 microM) occurs over a pH range of 7-10 with the optimum at 7.4 (Tris-HCl buffer). The optimum cleavage time is 3.5 h (22 degrees 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'-(phosphoglycolic acid). The products are consistent with the oxidative degradation of the deoxyribose ring of the DNA backbone, most likely by hydroxy radical.

PubMed Disclaimer

Cited by

Demonstration of a pronounced effect of noncovalent binding selectivity on the (+)-CC-1065 DNA alkylation and identification of the pharmacophore of the alkylation subunit.
Boger DL, Zarrinmayeh H, Munk SA, Kitos PA, Suntornwat O. Boger DL, et al. Proc Natl Acad Sci U S A. 1991 Feb 15;88(4):1431-5. doi: 10.1073/pnas.88.4.1431. Proc Natl Acad Sci U S A. 1991. PMID: 1847523 Free PMC article.
Possible chemical mechanisms underlying the antitumor activity of S-deoxyleinamycin.
Sivaramakrishnan S, Gates KS. Sivaramakrishnan S, et al. Bioorg Med Chem Lett. 2008 May 15;18(10):3076-80. doi: 10.1016/j.bmcl.2007.11.092. Epub 2007 Nov 28. Bioorg Med Chem Lett. 2008. PMID: 18068362 Free PMC article.
Torsional rigidities of weakly strained DNAs.
Fujimoto BS, Brewood GP, Schurr JM. Fujimoto BS, et al. Biophys J. 2006 Dec 1;91(11):4166-79. doi: 10.1529/biophysj.106.087593. Epub 2006 Sep 8. Biophys J. 2006. PMID: 16963514 Free PMC article.
Chemical cleavage of plasmid DNA by glutathione in the presence of Cu(II) ions. The Cu(II)-thiol system for DNA strand scission.
Reed CJ, Douglas KT. Reed CJ, et al. Biochem J. 1991 May 1;275 ( Pt 3)(Pt 3):601-8. doi: 10.1042/bj2750601. Biochem J. 1991. PMID: 2039439 Free PMC article.

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