Effect on DNA structure

FIGURE 12.16 The structures of ethidiutn bromide, acridine orange, and actinomycin D, three intercalating agents, and their effects on DNA structure. 

The nature of activating cations affects the substrate specificity of the enzyme, the optimum pH, and the maximum activity (3,4). DNase I introduces doublestrand breaks when Mn is used in place of Mg (3,5). The combination of Ca " and Mg " may shift the Mg -dependent nicking activity toward a doublestrand cutting activity. In contrast to the stability of DNase I in the presence of (with which the optimum temperature is 37°C), the enzyme is considerably more stable in the presence of Mn , losing no activity up to 60°C (6). The ability of transition metal ions such as Mn " and Co to promote double-strand cleavage is believed to be due at least in part to the stabilization and/or activation of a different enzyme structure (6) and also in part to the effect on DNA structure consequent to the binding of these metal ions (but not of Mg " or Ca ) to the N7 of the G base and an adjacent phosphate (5). 

A dramatic illustration of the effect of metal ions on DNA structure is seen in the STM images of DNA circles in the presence of either MgCl2 or ZnBr2 (46). The shape of the DNA circles of 168-base-pair (bp) length... 

Plastaras, J. P. Dedon, P. C. Marnett, L. J. Effects of DNA structure on oxopropenylation by the endogenous mutagens malondialdehyde and base propenal. Biochemistry 2002,41, 5033-5042. 

Crothers, D.M. (1993) Architectural elements in nucleoprotein structures. Curr. Biol. 3, 675-676. Stros, M. (2001) Two mutations of basic residues within the N-terminus of HMG-1 B domain with different effects on DNA supercoiling and binding to bent DNA. Biochemistry 40, 4769-4779. 

Hammermann, M., Steinmaier, C., Merlitz, H., Kapp, U., Waldeck, W., Chirico, G., and Langowski, J. (1997) Salt effects on the structure and internal dynamics of superhelical DNAs studied by light scattering and Brownian dynamics. Biophys. J. 73, 2674-2687. 

Under a variety of conditions, plasmid DNA undergoes a dramatic compaction in the presence of condensing agents such as multivalent cations and cationic polymers. Naked DNA coils, typically with a hydrodynamic size of hundreds of nanometers, after condensation it may become only tens of nanometer in size. Contrary to proteins which show a unique tertiary structure, DNA coils do not condense into unique compact structure. Cationic polymers execute their gene carrier function by their condensation effect on gene materials and, furthermore, their protection effect on DNA from nuclease digestion. Currently, the most widely used cationic polymers in research include linear or branched PEI (poly (ethyleneimine) (161-165), polypeptides such as PLL (poly-L-lysine) (166-169), PLA (poly-L-arginine) (170). 

Designing an isolation procedure for DNA requires extensive knowledge of the chemical stability of DNA as well as its condition in the cellular environment. Figures El3.1 and El3.2 illustrate several chemical bonds in DNA that may be susceptible to cleavage during the extraction process. The experimental factors that must be considered and their effects on various structural aspects of intact DNA are outlined below. 

Cellular anionic lipids have a twofold effect on DNA release from the lipo-plexes. They compensate the cationic lipid surface charge and eliminate the electrostatically driven DNA binding to the membrane interface, and they also disrupt the lipoplex structure and facilitate DNA departure into the solution by inducing formation of nonlamellar phases upon mixing with the lipoplex lipids. 

In addition to structure-activity relationships, dozens of useful tests have been developed for mutagenicity to germ cells and somatic cells and inferred carcinogenicity. The most straightforward means of testing for effects on DNA is an examination of DNA itself. This is normally difficult to do, so indirect tests are used. One useful test measures the activity of DNA repair mechanisms (unscheduled DNA synthesis) a higher activity is indicative of prior damage to DNA. 

In addition to studies dealing with structural chromosomal changes, effects on DNA were also investigated. Oral exposure to 1 g/kg/week of 2,3,7,8-TCDD or 1,2,3,7,8-PCDD for up to 6 months did not increase the formation of DNA adducts in Sprague-Dawley rats (Randerath et al. 1989). A single oral dose of 2,3,7,8-TCDD (25-100 g/kg) caused time-dependent increases in the induction of DNA single-strand breaks (and lipid peroxidation) in hepatic cells of Sprague-Dawley rats terminated within 3-14 days after the treatment (Wahba et al. 1989). 

These brief treatments of diverse osmotic effects on DNA are adequate to show that changes in the solute composition of the nucleoplasm and cytoplasm trigger a substantial number of effects on the structure and function of DNA and chromatin. These effects, which may be lethal if not reversed, are paired with regulatory responses that lead to changes in the amounts and activities of a wide range of enzymatic, transport, and gene regulatory proteins,... 

Chatterjee A, Holley WR. (1993) Computer-simulation of initial events in the biochemical-mechanisms of DNA-damage. Adv Radiat Biol 17 181-226. Goodhead DT, Leenhouts HP, Paretzke, HG, Terrisol M, Nikjoo H, Blaauboer 1C (1994) Track structure approaches to the interpretation of radiation effects on DNA. Radiat Prot Dosim 52 217-223. 

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