Fundamentals of Molecular Biology. Part 2: Molecular Genetic Mechanisms - A. N. Ogurtsov 2011

Basic Molecular Genetic Mechanisms
DNA Specificity

Nucleic Acids are polymers of NUCLEOTIDES that form a sugar-phosphate backbone with four alternating nucleic bases: A, T, C, G in DNA, and A, U, C, G in RNA (Figure 2).

DNA is a double helix consisting of two right-handed strands held together by Hydrogen Bonds between complementary Base Pairs A=T and C=G.

The predominant form of DNA is the B-form, although the A-form is also found, particularly in RNA-DNA and RNA-RNA Double helices, as well as the Z-form adopted by certain short DNA molecules. The cellular function of the Z-form remains unclear. Finally, synthetic polymers poly-A and poly-U can form triple helices.

Despite the high structural stability provided by multiple hydrogen and hydrophobic interactions, the canonical B-form of DNA exhibits considerable flexibility.

This property allows for the necessary conformational modifications of DNA upon protein binding to specific sites. Unlike protein α-helices, DNA lacks hydrogen bonds parallel to the

main axis, enabling the molecule to bend when associating with Proteins that direct DNA evolution (Figure 3).

The high flexibility of DNA ensures its tight packaging into nucleosomes of Chromatin—a DNA-protein complex that forms eukaryotic Chromosomes.

During METABOLISM/36.html">DNA Replication and Transcription, the two strands of The Double Helix must separate to allow the "inner" edges of the bases to pair with their respective complementary bases, thereby forming a new polynucleotide chain.

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Figure 2 - Cytology/cytology/92.html">SCHEMATIC Structure OF DNA: a - Van der Waals model; b - chemical bonding scheme in DNA

The process of unwinding the double helix and separating the two strands is known as DNA Denaturation (or melting).

Denaturation can be induced artificially (in vitro) by heating a DNA-containing solution. As the Temperature rises, thermal motion within the molecules increases, breaking the weak non-covalent bonds that hold the double helix together. The strands separate and drift apart due to electrostatic repulsion between the negatively charged sugar-phosphate backbones.

Figure 3 - DNA bending upon binding to the C-domain of the TATA-binding protein (TBP)

Near the denaturation temperature, a slight increase in the solution's temperature triggers a sharp, nearly simultaneous disruption of the multiple weak non-covalent interactions (which maintain the double helix) along the entire length of the DNA, resulting in an abrupt change in the solution's ultraviolet absorbance (Figure 4(a)).

The melting temperature, T_m, at which the two DNA strands separate, is determined by several factors.

Molecules with a higher C=G content melt at a higher temperature because C=G pairs, possessing three hydrogen bonds, are more stable than A=T pairs, which have only two. Consequently, the melting temperature can be used to estimate the concentration of C=G pairs (Figure 4(b)).

Figure 4 - DNA denaturation temperature: a - ultraviolet absorbance; b - dependence of the melting temperature T_m on the G=C pair concentration

The ion concentration in the solution also affects T_m, as the negatively charged phosphate groups of both strands are shielded by positive ions. Lowering the concentration of positive ions in the solution reduces the degree of shielding, which increases the repulsive forces between the sugar-phosphate backbones and lowers T_m.

Finally, drastic pH changes denature DNA even at low temperatures. At low pH values (acidic environment), nucleic acid bases become protonated and thus acquire a positive charge, repelling one another. At high pH values (alkaline environment), the bases lose protons, become negatively charged, and likewise repel each other.

The single-stranded DNA molecules resulting from denaturation form randomly coiled structures lacking any definite conformation.

Conversely, (1) lowering the temperature, (2) increasing the ionic concentration, or (3) neutralizing the pH induces the spontaneous renaturation (or "annealing") of the two DNA strands back into a double helix.

Crucially, however, non-complementary DNA strands do not renature, remaining instead as random coils. Even more importantly, they do not interfere with two complementary DNA strands finding each other in solution and renaturing.

The processes of DNA Denaturation and renaturation (melting and annealing) form The basis of DNA Hybridization—a technique used to determine the sequence identity of two DNA samples and to detect and isolate specific DNA molecules in a solution containing various DNA sequences.