DNA Can Undergo Reversible Strand Separation

During replication and transcription of DNA, the strands of the double helix must separate to allow the internal edges of the bases to pair with the bases of the nucleotides to be polymerized into new polynucleotide chains. In later sections, we describe the cellular mechanisms that separate and subsequently reassociate DNA strands during replication and transcription. Here we discuss factors influencing the in vitro separation and reassociation of DNA strands.

The unwinding and separation of DNA strands, referred to as denaturation, or "melting," can be induced experimentally by increasing the temperature of a solution of DNA. As the thermal energy increases, the resulting increase in molecular motion eventually breaks the hydrogen bonds and other forces that stabilize the double helix; the strands then separate, driven apart by the electrostatic repulsion of the negatively charged deoxyribose-phosphate backbone of each strand. Near the denaturation temperature, a small increase in temperature causes a rapid, near simultaneous loss of the multiple weak interactions holding the strands together along the entire length of the DNA molecules, leading to an abrupt change in the absorption of ultraviolet (UV) light (Figure 4-6a).

The melting temperature Tm at which DNA strands will separate depends on several factors. Molecules that contain a greater proportion of G-C pairs require higher temperatures to denature because the three hydrogen bonds in G-C pairs make these base pairs more stable than A-T pairs, which have only two hydrogen bonds. Indeed, the percentage of G-C base pairs in a DNA sample can be estimated from its Tm (Figure 4-6b). The ion concentration also influences the Tm because the negatively charged phosphate groups in the

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