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DNA Replication and Repair

DNA Replication & DNA Repair: Comprehensive Study Notes

Part 1: Pattern of DNA Replication

Base-pairing Enables DNA Replication

Fundamental to DNA replication is the principle of base-pairing, where each existing strand of DNA serves as a template for the synthesis of a new, complementary strand. This process strictly adheres to specific pairing rules:

  • Adenine (A) always pairs with Thymine (T).

  • Cytosine (C) always pairs with Guanine (G).

Models for DNA Replication

Historically, three hypothetical models were proposed to explain how DNA replicates:

  • (A) Semiconservative Replication: Predicts that each new DNA molecule will consist of one original (parental) strand and one newly synthesized strand. After one generation, all DNA molecules would be hybrids, containing both heavy and light isotopes if initially grown in heavy medium and then transferred to light medium.

  • (B) Dispersive Replication: Suggests that each new DNA molecule would contain a mixture of parental and newly synthesized DNA on both strands, interspersed throughout the molecule. After one generation, all DNA molecules would be hybrids, but their density would be uniformly intermediate.

  • (C) Conservative Replication: Postulates that the original parental DNA molecule remains entirely intact, serving as a template for the synthesis of an entirely new daughter DNA molecule. After one generation, there would be two distinct DNA molecules: one purely parental ({}^{15} ext{N}) and one purely new ({}^{14} ext{N}), resulting in two distinct density bands.

Meselson-Stahl Experiment: Confirming Semiconservative Replication

The Meselson-Stahl experiment conclusively ruled out the conservative and dispersive models, providing strong evidence for semiconservative replication.

Methodology:

  1. Isotope Labeling: Escherichia coli (E. ext{coli}) bacteria were grown for many generations in a medium containing a heavy isotope of nitrogen, {}^{15} ext{N}. This caused all their DNA to incorporate {}^{15} ext{N} and thus be denser.

  2. Density Gradient Centrifugation: DNA was extracted from the bacteria and subjected to centrifugation in a cesium chloride ( ext{CsCl}) density gradient. This technique separates DNA molecules based on their density: {}^{15} ext{N}-DNA bands at a higher density (closer to the bottom of the tube), while {}^{14} ext{N}-DNA (from bacteria grown in a lighter {}^{14} ext{N} medium) bands at a lower density (closer to the top).

  3. Experimental Procedure:

    • Control 1: {}^{14} ext{N}-DNA extracted from E. coli grown in {}^{14} ext{N} medium, forming a light band.

    • Control 2: {}^{15} ext{N}-DNA extracted from E. coli grown in {}^{15} ext{N} medium, forming a heavy band.

    • Step 1: {}^{15} ext{N}-labeled E. coli were transferred to a {}^{14} ext{N} medium.

    • Step 2 (First Generation, 30 minutes): After one round of replication in {}^{14} ext{N} medium, DNA was extracted and centrifuged. The result was a single band of intermediate density.

    • Step 3 (Second Generation, 60 minutes): The cells were allowed to replicate a second time in {}^{14} ext{N} medium. DNA was again extracted and centrifuged. This time, two bands were observed: one intermediate density band and one light density band.

Results and Conclusion:

  • The presence of a single intermediate band after the first generation was inconsistent with the conservative model (which would predict heavy and light bands) and dispersive model (which would predict only one intermediate band, but without separation into light and intermediate in the second generation). It supported the semiconservative model, where each new DNA molecule was a hybrid of {}^{15} ext{N} and {}^{14} ext{N}.

  • The presence of two bands (one intermediate and one light) after the second generation directly supported the semiconservative hypothesis. The intermediate band represented hybrid molecules, while the light band represented fully {}^{14} ext{N} DNA molecules synthesized from the {}^{14} ext{N} template strands of the first-generation hybrids. This pattern is precisely what semiconservative replication predicts, as shown in Figure 6.20

Therefore, DNA replication is a semiconservative process.

Part 2: DNA Replication Mechanism

DNA Synthesis Begins at Replication Origins

DNA synthesis does not start randomly but at specific sites called replication origins. These are particular nucleotide sequences recognized by initiator proteins. These proteins bind to the origin, locally separating the two DNA strands, creating a