DNA replication is the process by which a cell duplicates its genetic material, ensuring subsequent cells receive an accurate copy of the genome. Before this mechanism was understood, scientists debated how the double-stranded parent molecule distributed its genetic information to the two new daughter molecules. The complementary structure of the DNA double helix suggested a template-based mechanism, but the exact outcome regarding old versus new material remained unknown. This led to the proposal of three distinct theoretical models, each predicting a different fate for the original DNA strands after one round of duplication.
Defining the Replication Hypotheses
The three initial hypotheses proposed different methods for incorporating the two strands of the parent DNA molecule into the new double helices. In the conservative model, the entire original DNA molecule remained fully intact after replication. The two parent strands would stay together, acting as a guide to synthesize a completely new double helix composed only of new material. This process would yield one old molecule and one entirely new molecule following a single round of duplication.
The semiconservative model proposed that the two strands of the parent helix would first separate. Each original strand would then serve as a template for synthesizing a new, complementary strand, resulting in two new DNA molecules. Each daughter molecule would be a hybrid, consisting of one strand from the original parent molecule and one newly synthesized strand.
The dispersive model suggested that the original DNA molecule would be broken down into segments during replication. The resulting daughter molecules would be formed as a patchwork, with each individual strand containing a mixture of both old and newly synthesized DNA segments. Under this model, the old material would be scattered randomly throughout the two new helices, meaning neither the original helix nor its individual strands would be preserved.
The Meselson-Stahl Experiment
The question of which model was correct was settled by the landmark experiment performed by Matthew Meselson and Franklin Stahl in 1958. Their methodology relied on using nitrogen isotopes to physically tag the parent DNA. They grew Escherichia coli bacteria in a medium containing the heavy isotope, nitrogen-15 (\(^{15}\)N), until the DNA was fully labeled. This heavy DNA was distinguished from normal, light DNA (labeled with \(^{14}\)N) using density gradient centrifugation, which separates molecules based on mass.
The critical step involved transferring the \(^{15}\)N-labeled bacteria into a medium containing only the light isotope, \(^{14}\)N, allowing them to undergo DNA replication. After one generation, the researchers analyzed the DNA and observed only a single band with an intermediate density. This hybrid result immediately ruled out the conservative model, which predicted two separate bands: one heavy (\(^{15}\)N/\(^{15}\)N) and one light (\(^{14}\)N/\(^{14}\)N).
However, the intermediate band was consistent with both the semiconservative and the dispersive models. To distinguish between the two, the bacteria were allowed to replicate for a second generation in the light \(^{14}\)N medium. The DNA was again isolated and centrifuged, revealing two distinct bands. One band was still the intermediate hybrid density, and the second band was entirely light density (\(^{14}\)N/\(^{14}\)N).
This result provided the evidence needed: the presence of both hybrid and light DNA in equal amounts only fit the semiconservative model. The dispersive model would have predicted that all DNA molecules would remain a single, increasingly lighter intermediate band. The data confirmed that each parent strand separated and remained intact as a template, establishing that the mechanism of DNA replication is semiconservative.
Semiconservative Replication in Action
Once the semiconservative model was established, attention shifted to the cellular machinery that carries out the copying process. Replication begins at specific points where the double helix must be unwound and separated. The enzyme helicase breaks the hydrogen bonds between base pairs, creating the Y-shaped replication fork. As the DNA unwinds, topoisomerases work ahead of the fork to relieve the torsional stress, or supercoiling, that would otherwise halt the process.
The actual synthesis of the new strand is carried out by DNA polymerase, an enzyme that can only add new nucleotides in the 5′ to 3′ direction. This directional constraint means that the two template strands at the replication fork are synthesized differently. The leading strand template runs in the 3′ to 5′ direction, allowing the new strand to be synthesized continuously in a single, long piece, moving toward the advancing replication fork.
The other template strand, the lagging strand, runs in the 5′ to 3′ direction, forcing the DNA polymerase to work discontinuously, moving away from the fork. This strand is synthesized as a series of short segments known as Okazaki fragments, each requiring an RNA primer to begin synthesis.
Lagging Strand Synthesis
Once the Okazaki fragments are completed, the RNA primers are removed and replaced with DNA. A final enzyme, DNA ligase, seals the gaps between the fragments to create a continuous strand. This precise, semiconservative method ensures that the genetic information is duplicated with high fidelity.