Double-stranded cleavage of pBR322 by a diiron complex via a "hydrolytic" mechanism.

Abstract

Treatment of plasmid pBR322 with Fe2-(HPTB)(OH)(NO3)4(HPTB =N,N,N',N '-tetrakis(2-benzimidazolylmethyl The complex Fe2(HPTB)(OH)(NO3)4 was synthesized according to published procedures (20). This complex has been shown to interact with H202 to form a 1:1 adduct (20).Plasmid pBR322 was purchased from Promega and transformed into Escherichia coli strains JM109 and HB101 by using CaCl2 (22). The cells were grown in Luria-Bertani broth containing tetracycline, amplified with chloramphenicol, and harvested; the plasmid was purified according to published procedures (23). Purity and quantity of DNA were determined by measuring the A26o/A280 ratio, using a Hoefer fluorometer and gel electrophoresis.Linearization of pBR322 with Fe2(HPTB)(OH)(NO34. The cleavage of pBR322 by Fe2(HPTB)(OH)(NO3)4/H202 was accomplished by mixing (in order) 1 ,ul of (0.15 mg/ml) pBR322, 1 ,ul of 0.1 M Tris HCl (pH 8.0) buffer, 1,ul (0.1 mM) of Fe2(HPTB)(OH)(NO3)4, 6 ,l of H20, and 1 ,ul of 9.0 mM H202 at 25°C. Immediately after mixing, the DNA solution was subjected to electrophoresis on a 1% agarose gel in TAE (40 mM Tris acetate/i mM EDTA) (24) and stained with ethidium bromide solution at 0.5 pg/ml to observe the cleaved DNA products. The amount of DNA was quantitated by photographing the fluorescence under UV light and analyzing the band intensities with a Pharmacia densitometer. The fluorescence intensities were corrected for the different binding affinities ofethidium bromide to form I vs. forms II and III (25). Lengthening the reaction time did not affect the amounts of nicked and linearized pBR322. The linearized pBR322 was recovered from the agarose gel by electroelution ofthe excised gel fragment, followed by phenol/chloroform extraction and precipitation with 3.0 M NaOAc and ethanol (24).Control experiments with Fe(EDTA)/H202 were done under conditions identical to those of Fe2(HPTB)(OH)(NO3)4/ Abbreviations:

Efficient Increase of DNA Cleavage Activity of a Diiron(III) Complex by a Conjugating Acridine Group

“…Two Fe III complexes with high hydrolytic activity in DNA cleavage have been reported and both of them are binuclear. The first complex Fe 2 (HPTB), described by Schnaith, Que and co-workers in 1994 [25] showed a surprising reactivity: in the presence of the complex at a concentration of 10 µ at 25°C and a www.eurjic.orgpH of 8.0, the supercoiled form was completely degraded immediately after mixing. However, the cleavage system was very complicated because high activity depended on "oxidative" conditions, i.e.…”

“…The earlier two diiron(III) complexes Fe 2 (DTPB) and Fe 2 (HPTB), described respectively by Liu [23] and by Que, [25] have shown the yielding linearised DNA can be quantitatively relegated by confirming the occurrence of a hydrolytic mechanism. In our cases, the linearised DNA has also been relegated by T4 ligase (Figure 6), although the relegation is not complete.…”

“…Among the physiologically relevant metal ions, iron has been much less studied in the hydrolytic reaction [23][24][25] due to the fact that Fe-dependent DNA cleavage is often involved in oxygen free radical processes such as the Fenton reaction. In fact, purple acid phosphatases (PAP), comprising diiron active centers can catalyse the hydrolysis of certain phosphate esters, including nucleoside di-and triphosphates and aryl phosphates under weakly acidic conditions.…”

Synthesis, crystal structure, magnetic property, and nuclease activity of a binuclear iron(III) complex

“…Among the active transition metal species, dinuclear, and polynuclear complexes generally give higher cleavage rates provided that the ligand holds the metal centers in an appropriate geometry [2][3][4][5][6][7][8][9]. Compared with mononuclear complexes, dinuclear complexes have higher activity as a result of the cooperative interaction of metal ions in stabilizing the transition state of phosphodiester cleavage.…”

“…Compared with mononuclear complexes, dinuclear complexes have higher activity as a result of the cooperative interaction of metal ions in stabilizing the transition state of phosphodiester cleavage. Dinuclear oxo-bridged complexes have proved to be effective chemical nucleases [8,9].…”