Chemical nuclease

Sigman DS (1986) Nuclease activity of 1,10-phenanthroline-copper ion. Acc Chem Res 19 180-186 Sigman DS (1990) Chemical nucleases. Biochemistry 29 9097-9105... 

Sigman DS, Chen CC (1990) Chemical nucleases new reagents in molecular biology. Annu Rev Bio-chem 59 207-236... 

Sigman DS, Mazumder A, Perrin DM (1993a) Chemical nucleases. Chem Rev 93 2295-2316 Sigman DS, BruiceTW, Mazumder A, Sutton CL (1993b) Targeted chemical nucleases. Acc Chem Res 26 98-194... 

Chemical nucleases provide detailed information concerning the interactions of regulatory proteins with DNA (12) and have recently been applied to intact transcription complexes. High-resolution probes such as hydroxyl radical-FeEDTA (192), as well as dimethyl sulfate (DMS) and DNAsel, were used to deconvolute the interactions of MerR, Hg-MerR, and RNA polymerase with Pr, the DNA sequences in the regulatory region (the promoter) of the mercuric ion responsive genes (145). These studies provide a structural map, also known as a footprint , of the interactions of both proteins with the DNA in complexes isolated at different stages in the activation process and provide structural information on the physical role Hg-MerR in the activation mechanism. Several of the kinetic inter-... 

Figure 19. Structures of the chemical nucleases methidium propyl EDTA-Fe(II) (MPE) (bottom) and copper-5-phenyl-o-phenanthroline (phenyl-CuOP) (top).

Figure 20. Schematic mechanism of mercuric ion-responsive activation of transcription. A distortion of the DNA at the center of the Hg-MerR binding site, deduced by hyperreactivity of chemical nucleases, is represented by V-shaped dashed lines. This distortion is present when MerR is associated with Hg(II) in both the presence and absence of RNA polymerase, as shown. Nucleases that demonstrate hyperreactivity are MPE and phenyl-CuOP (65). Bars represent DNA sequences of mer genes joined rectangles represent the dimeric protein MerR, shown associated with Hg(II) in complexes c and d. Shaded ellipsoids represent the protein RNA polymerase.

As a subject for mechanistic study, Blm is an enormously important molecule. It is an effective antitumor agent and a model for an effective metallodrug. Furthermore, it is a highly efficient reagent for the generation of DNA double strand breaks. As such, it has also become the prototype for the design of synthetic chemical nucleases involving redox-active metal sites. ... 

The observation of 4 -hydroxylated abasic site 23 allowed to propose the mechanism summarized in Fig. 7 in association to the observation of similar oxidation products previously characterized with Fe-bleomycin or neocarzi-nostatin (4,7). It results in the initial formation of a C4 radical 6 due to H4 abstraction by activated Cu(phen)2. Then a C4 -hydroxylated compound is probably formed. It allows the release of the nucleobase and the formation of 4 -hydroxylated abasic site 23 that is not associated to DNA cleavage. This site was trapped by the authors as a pyridazine after reaction with hydrazine followed by an enzymatic digestion to nucleosides. Oyushi and Sugiyama proposed that a C4 carbocation was involved as intermediate in the reaction, as for Cl -DNA oxidation performed by activated Cu(phen)2. This hypothesis needs, however, to be confirmed since other evolutions of the C4 radical 6, producing also C4 -hydroxylated site, have been proposed with other chemical nucleases (4). However, in the case of DNA oxidation by activated Cu(phen)2 this oxidation pathway seemed minor when compared to the pathway leading to the formation of phosphoglycolate fragment 11. 

The mechanisms of oxidative DNA damage reported in vitro with these two chemical nucleases illustrate the diversity of the processes that one may consider for the study of in vivo DNA damage. 

Scheme 1. Chemical structures involved in DNA breaks generated by the chemical nuclease Mn-TMPyP/KHS05.

Pratviel G, Bemadou J, Meunier B. Carbon-hydrogen bonds of DNA sugar units as targets for chemical nucleases and drugs. Angetv Chem Int Ed Engl. 1995 34 746-769. 

Another class of promising new generation nucleases includes artificial and semiartificial nucleases. Chemical nucleases are typical artificial nucleases that make use of redox-active compounds (e.g., phenanthroline-copper and ferrous-EDTA) as the cutter and an attached oligonucleotide as the site-specific recognition module. Although the importance of chemical nucleases and the potential for their applications are bound to grow, this chapter focuses on some of the classical enzyme nucleases that are most widely used in recombinant DNA technology. 

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