Divyan Bavan
Our knowledge of DNA structure and replication has enabled a revolution in genetic approaches to biology. While Watson and Crick’s seminal paper on the structure of DNA is the most recognized contribution in this field, it is just one in a long list of important experiments that elucidated the properties of DNA. These breakthroughs came over a span of decades—from Miescher’s discovery of nucleic acids to the discovery of the lagging strand in DNA replication. The common string, however, is experimental evidence, which introduces, proves, and updates hypotheses given by scientists. These experiments underpin all the discoveries in DNA structure and replication.
The study of the former started in 1869, when Friedrich Miescher isolated nuclein—which is now known as DNA—from pus cells. Following this discovery, several advancements were made in our understanding of the composition of DNA. Through the purification, hydrolysis, and separation of DNA, Phoebus Levene was able to identify the main components of DNA: the four nucleotides (Voet and Voet, 2011). Given this evidence, he and other scientists proposed the tetranucleotide hypothesis. This theory stated that DNA was a polymer that had the same repetition of four nucleotides, and thus had the same proportion of each nucleotide and no genetic role. To determine if this hypothesis was true, more evidence was needed. Erwin Chargaff provided it. He used paper chromatography and UV spectrophotometry to quantify the proportions of each nucleotide in DNA, allowing him to see that bases did not come in equal amounts, disproving the tetranucleotide hypothesis (Chargaff, 1948). However, Chargaff found that the nucleotides did come in proportional pairs, with adenine and thymine having roughly equal amounts, and the same with cytosine and guanine. This example of experimental evidence shows how it can change our understanding of a topic completely, but it can also spawn new areas to explore. With Chargaff’s Rule, scientists were able to try and elucidate the chemical structure of DNA. While this was eventually produced by Watson and Crick, scientists did not immediately succeed. Linus Pauling theorized that DNA was a triple helix with phosphates on the inside and bases on the outside (Pauling and Corey, 1953). This wasn’t correct, and experimental evidence was needed to correct their theory. This came in the form of X-ray diffraction data. Rosalind Franklin and Maurice Wilkins had used this technique to collect data about the positions of nucleotides in DNA, discovering that the bases stacked on the inside of the helix (Voet and Voet, 2011). With access to this data, Watson and Crick eventually came to the double-helix model of DNA. Again, this shows how experimental evidence can quickly change theories and push them in the right direction. However, it can also add to theories without modifying existing ones. In an experiment by Andrew Wang and Alexander Rich, it was shown that the self-complementary DNA sequence CGCGCG had a left-handed spiral with a deep minor groove and no major groove (Voet and Voet, 2011). This is fundamentally different from standard Watson-Crick structures, which have a right-handed spiral with both a minor and major groove. This was shown to be a product of a complete flip in the bases. It is an example of how experiments do not only prove or disprove theories, but can also add depth to them.
A similar story has unfolded with DNA replication, with key experiments paving the way for our understanding. As predicted by Watson and Crick, the base-pairing mechanisms provides a direct way to transfer information in duplication events (Watson and Crick, 1953). However, understanding how this process worked took several experiments, the first of which was the Meselson-Stahl experiment.
The first generation of bacteria, grown in N15 media, produced a band corresponding to “heavy” DNA in density gradient centrifugation. The second generation produced one band: an intermediate between N14 and N15 DNA. This ruled out conservative replication, as if it were true, there would be both a heavy and light band. However, there was still the possibility for dispersive replication, as different segments could be used and create an intermediate. To rule this out, a third generation was produced, which formed a light N14 band and an intermediate band. This ruled out the dispersive model, as that would have produced a single band closer to the light band. Therefore, Meselson and Stahl were able to show that DNA replication is semiconservative.
While this experiment elegantly showed the core idea behind DNA replication, it did not give insight into how the replication happened. This came instead with a discovery by Arthur Kornberg. With E. coli lysate, he was able to discover an enzyme which would incorporate a radioisotope of thymidine triphosphate into DNA, synthesizing a new strand. He called this enzyme DNA polymerase I—the enzyme behind strand synthesis (Voet and Voet, 2011). This shows how experimental evidence adds depth to theories, creating more details to have a better picture of the system. However, although this protein was a compelling candidate for the main replicative machine, it was later shown that DNA polymerase III is the primary replicative protein. This was shown by analyzing E. coli samples with little DNA polymerase I expression, with the evidence indicating another protein doing a large amount of replication. Even then, more experiments needed to be done to finalize the process. This was accomplished by Reiji and Tsuneko Okazaki, who labeled replicating DNA with [3H]thymidine (Voet and Voet, 2011). Their experiments showed that DNA was being produced in short fragments and linked together, as after the E. coli were transferred to an unlabelled medium, the labeled DNA appeared continuous. This uncovered Okazaki fragments—products of the lagging strand in DNA replication. Along with the discoveries of topoisomerase, single-strand binding proteins, DNA helicase, and other proteins in the replisome, these findings greatly elucidated the mechanism behind DNA replication, showing the power of experimental evidence in quickly characterizing a scientific process.
Through these experiments, scientists were able to discover the details behind DNA structure and replication. They have led to the incredible advances in genomic medicine, research, and our overall understanding of life. However, it extends far beyond that. The lessons learned from the history of these experiments offer a view into how science should be conducted—coming up with new ideas when evidence presents itself but being ready to update or discard those ideas when more evidence comes out. This process is central to scientific discovery and underpins all the discoveries that have been and will be made.