DNA Can be Reversibly Denatured & Specifically Renatured, Both in the Test Tube & in Living Cells
المؤلف:
Peter J. Kennelly, Kathleen M. Botham, Owen P. McGuinness, Victor W. Rodwell, P. Anthony Weil
المصدر:
Harpers Illustrated Biochemistry
الجزء والصفحة:
32nd edition.p350-351
2025-09-06
464
In the laboratory the double-stranded structure of DNA can be separated, or denatured into its two component strands in solution by: increasing temperature, decreasing solution salt concentrations, adding chaotropic agents, which can form competing H-bonds with the individual deoxynucleotide bases, or often experimentally, by a combination of all three treatments. Under such conditions not only do the two stacks of bases pull apart, but the bases themselves unstack while remaining connected within the now, two single-stranded polymers, that are connected by their phosphodiester backbones. Concomitant with the denaturation of the DNA molecule into two single strands is an increase in the optical absorbance in the ultraviolet light spectrum (260 nm) of the purine and pyrimidine bases of each strand; this phenomenon is referred to as hyperchromicity of denaturation. Because of the combined strength of base stacking and the H-bonding between the complementary bases in each strand, the double-stranded DNA molecule exhibits properties of a rigid rod. Thus, native double stranded DNA in solution is an extremely viscous material. However, on denaturation, DNA solutions lose their viscosity.
The strands of a given molecule of double-stranded DNA separate over a temperature range. The midpoint of the measured DNA denaturation is called the melting temperature, or Tm . The Tm is influenced by the base composition of the DNA and by the salt concentration or other components of the solution . DNA rich in G–C pairs melts at a higher temperature than DNA rich in A–T pairs, due to differences in hydrogen bond content and base stacking, as discussed earlier. A 10-fold increase of monovalent cat ion concentration significantly increases the Tm by neutralizing the intrinsic interchain repulsion between the highly negatively charged phosphates of the phosphodiester backbone of each DNA strand. For example, an increase of NaCl concentration from 0.01 to 0.1 M increases Tm by 16.6°C. By contrast, chaotropes such as urea (NH2CONH2 ; see Figure 1) and formamide (CH3NO) can efficiently form H-bonds with the nucleotide bases, which destabilizes H-bonding between bases. Such solution conditions will lower the Tm. Chaotrope addition allows the strands of DNA or complementary DNA–RNA, and intramolecular RNA-RNA hybrids to be separated at much lower temperatures. Lower temperatures minimize phosphodiester bond breakage and chemical damage to nucleotides that can occur on extended incubation in solution. In living cells, both DNA denaturation and renaturation occurs naturally during the processes of DNA replication, DNA recombination, DNA repair, and DNA gene transcription. In all of these instances DNA strand separation and renaturation is mediated through the action of specific nucleic acid binding proteins and various enzymes, in combination with thermal and/or chemical energy supplied via ATP hydrolysis.
Fig1. Reactions and intermediates of urea biosynthesis.The nitrogen-containing groups that contribute to the formation of urea are shaded. Reactions 1 and 2 occur in the matrix of liver mitochondria and reactions 3 , 4 , and 5 in liver cytosol. CO2 (as bicarbon ate), ammonium ion, ornithine, and citrulline enter the mitochondrial matrix via specific carriers (see red dots) present in the inner membrane of liver mitochondria.
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