Nucleic Acid Chemistry:- Double-Helical DNA and RNA Can Be Denatured
Solutions of carefully isolated, native DNA are highly viscous at pH=7.0 and room temperature (25 C). When such a solution is subjected to extremes of pH or to temperatures above 80 OC, its viscosity decreases sharply, indicating that the DNA has undergone a physical change. Just as heat and extremes of pH denature globular proteins, they also cause denaturation, or melting, of double-helical DNA. Disruption of the hydrogen bonds between paired bases and of base stacking causes unwinding of the double helix to form two single strands, completely separate from each other along the entire length or part of the length (partial denaturation) of the molecule. No covalent bonds in the DNA are broken (Fig. 8–29).
Renaturation of a DNA molecule is a rapid one-step process, as long as a double-helical segment of a dozen or more residues still unites the two strands. When the temperature or pH is returned to the range in which most organisms live, the unwound segments of the two strands spontaneously rewind, or anneal, to yield the intact duplex (Fig. 8–29). However, if the two strands are completely separated, renaturation occurs in two steps. In the first, relatively slow step, the two strands “find” each other by random collisions and form a short segment of complementary double helix. The second step is much faster: the remaining unpaired bases successively come into register as base pairs, and the two strands “zipper” themselves together to form the double helix.
The close interaction between stacked bases in a nucleic acid has the effect of decreasing its absorption of UV light relative to that of a solution with the same concentration of free nucleotides, and the absorption is decreased further when two complementary nucleic acids strands are paired. This is called the hypochromic effect. Denaturation of a double-stranded nucleic acid produces the opposite result: an increase in absorption called the hyperchromic effect. The transition from double-stranded DNA to the single-stranded, denatured form can thus be detected by monitoring the absorption of UV light.
FIGURE 8–29 Reversible denaturation and annealing (renaturation) of DNA.
Viral or bacterial DNA molecules in solution dena ture when they are heated slowly (Fig. 8–30). Each species of DNA has a characteristic denaturation temperature, or melting point (tm): the higher its content of G≡C base pairs, the higher the melting point of the DNA. This is because G≡C base pairs, with three hydrogen bonds, require more heat energy to dissociate than A≡T base pairs. Careful determination of the melting point of a DNA specimen, under fixed conditions of pH and ionic strength, can yield an estimate of its base composition. If denaturation conditions are carefully controlled, regions that are rich in A≡T base pairs will specifically denature while most of the DNA remains
FIGURE 8–30 Heat denaturation of DNA. (a) The denaturation, or melting, curves of two DNA specimens. The temperature at the midpoint of the transition (tm) is the melting point; it depends on pH and ionic strength and on the size and base composition of the DNA. (b) Relationship between tm and the G≡C content of a DNA.
FIGURE 8–31 Partially denatured DNA. This DNA was partially de natured, then fixed to prevent renaturation during sample preparation. The shadowing method used to visualize the DNA in this electron micrograph increases its diameter approximately fivefold and obliterates most details of the helix. However, length measurements can be obtained, and single-stranded regions are readily distinguishable from double-stranded regions. The arrows point to some single-stranded bubbles where denaturation has occurred. The regions that denature are highly reproducible and are rich in A≡T base pairs.
double-stranded. Such denatured regions (called bubbles) can be visualized with electron microscopy (Fig. 8–31). Strand separation of DNA must occur in vivo during processes such as DNA replication and transcription. As we shall see, the DNA sites where these processes are initiated are often rich in A≡T base pairs. Duplexes of two RNA strands or of one RNA strand and one DNA strand (RNA-DNA hybrids) can also be denatured. Notably, RNA duplexes are more stable than DNA duplexes. At neutral pH, denaturation of a double helical RNA often requires temperatures 20 C or more higher than those required for denaturation of a DNA molecule with a comparable sequence. The stability of an RNA-DNA hybrid is generally intermediate between that of RNA and that of DNA. The physical basis for these differences in thermal stability is not known.
