BIOCHEMISTRY - Textbook - Ostapchenko L. I. - 2012

Chapter 9. NUCLEIC ACIDS

9.4. Structure of Nucleic Acids

9.4.4. DNA Denaturation and Renaturation

Investigating the mechanisms of DNA Denaturation and renaturation is crucial for understanding its Structure and biological role. Since the two DNA strands are held together solely by non-covalent (hydrogen) bonds and base-stacking interactions between adjacent bases, physical or Chemical factors can cause a DNA molecule to dissociate into separate strands and adopt an unordered random-coil conformation. This process of DNA strand Separation is referred to as denaturation, or melting, of The Double Helix.

Double-stranded helical DNA in solution is readily disrupted by heating to temperatures near 100 °C, as well as by increasing the solution pH to a level that disrupts the Hydrogen Bonds between bases. Other factors (such as mono- and divalent cations, Polyamines, and Proteins) influence denaturation by partially or completely neutralizing the negatively charged phosphate groups within the nucleic acid molecule.

These factors disrupt the native double-helical conformation of the DNA molecule, rendering the arrangement of purine and pyrimidine NUCLEOTIDES less ordered and triggering drastic changes in many of its physical properties. A particularly significant change is the increase in ultraviolet absorbance (with a maximum near 260 nm, characteristic of each base). This rise in optical density upon DNA denaturation is known as the hyperchromic effect.

The Temperature or pH range over which DNA denaturation occurs is very narrow; characteristic parameters of this process include the melting temperature (T_m) or melting pH (pH_m), which represent the midpoint of the temperature or pH range at which DNA strand separation takes place (Fig. 9.14). The rising portion of the curve corresponds to the specific temperature range (Fig. 9.14, A) or pH range (Fig. 9.14, B) over which the double-stranded DNA completely dissociates. Temperature and pH are reproducible Physical Properties of the DNA molecule under specific conditions.

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Fig. 9.14. Denaturation curves of typical double-stranded DNA with increasing temperature (A) and medium pH (B)

Because disrupting the two hydrogen bonds of A-T pairs requires less energy than breaking the three hydrogen bonds of G-C pairs, the denaturation temperature or pH depends on the Nucleotide Composition of the DNA. The higher the G-C content, the higher the T_m or pH_m (Fig. 9.15). During denaturation, A-T-rich regions of DNA melt first.

Fig. 9.15. DNA melting curves depending on the nucleotide COMPOSITION OF THE molecules

Denaturation is a reversible process. Under appropriate conditions, two separated complementary DNA strands can reform the double helix.

This phenomenon, known as renaturation, reassociation, or annealing, occurs when temperature or pH is lowered. Upon slow cooling or a decrease in pH, the previously disordered single DNA strands reform a double helix through base pairing, causing the molecule to renature. DNA molecules with a homogeneous nucleotide composition renature at a faster rate.

The ability of DNA to renature is used to obtain data on the quantity of homologous sequences in test nucleic acid samples. This is achieved using a method based on The formation of hybrid duplexes (heteroduplexes) between DNA and RNA, or between two DNA strands isolated from different organisms. Hybridization studies have revealed a clear pattern: the greater the phylogenetic affinity between two species, the higher the capacity of their DNAs to form hybrids. The molecular hybridization method makes it possible to study the DNA relatedness of various species and draw Conclusions about their genetic similarity.