It was not that long ago that simple diseases like strep and tuberculosis killed many individuals. However, with the introduction of technology into the laboratory, our knowledge of previously obscure scientific areas has begun to come to light. Individuals in the fields of genetics, pathology, and immunology have made discoveries that have taught us how cells work, and thus how to protect them from invasion and destruction.

Geneticists study DNA, the genetic makeup that codes for all traits and characteristics in all living things. Studying genes can help us understand why cells will act in a specific way, and can help us predict how they will act in the future. Though Gregor Mendel provided us with wonderful information about genetics (considering his very limited resources and background), until the nineteenth century, no one knew exactly how traits were determined. In 1869, Friedrich Miescher was the first to isolate and identify genetic material. In 1953, James Watson and Frances Crick proposed the double helix model for DNA that is still in use.

What, exactly, does DNA do? DNA directs the synthesis of proteins and the development of cells, and is the substance that is copied to ensure similar processes of development in offspring (Kornberg, A). DNA carries specific instructions for specific tasks, and exact copies of these instructions are made so the information can be used “again and elsewhere.” Now that we have established the function of DNA, we can explore how the replication of DNA occurs on an enzymatic level. To do this, we must first understand the molecular structure of a DNA molecule.

A DNA molecule is composed of chains of nucleic acids. Nucleic acids are comprised of a five-carbon sugar, a phosphate group, and a nitrogenous base. There are four possible nitrogenous bases that can be found in a nucleic acid: two purines, adenine and guanine, and two pyrimidines, cytosine and thymine. Studies have shown that the purine content of a DNA molecule always equals the pyrimidine content; that is, in a DNA molecule, the number of nucleic acids containing a purine equals the number of nucleic acids containing a pyrimidine. However, the number of adenines differs from the number of guanines among the purines, and the number of cytosines differs from the number of thymines among the pyrimidines (Kornberg, A).

In Watson and Crick’s double-stranded model of DNA, they showed how the oxygen and hydrogen atoms of a purine molecule forms hydrogen bonds with the hydrogen, oxygen and nitrogen atoms of a pyrimidine molecule, thus accounting for the equal ratio of purines to pyrimidines. The purine and pyrimidine bases of one chain are bonded to the pyrimidine and purine bases of the complementary chain in a helical array.  Watson and Crick used X-ray crystallography to show that the distance between the two chains in the model matches their estimated value for the length of the hydrogen bonds between purines and pyrimidines (Watson, J. and Crick, F.).

In 1959, Arthur Kornberg, along with Severo Ochoa, won the Nobel Prize in Physiology or Medicine for their discovery of the mechanisms of the synthesis of DNA and RNA. Kornberg spent decades isolating and purifying the enzymes that run the cell, leading him to discover the enzyme that catalyzes the synthesis of DNA, which we now know as DNA Polymerase I (Kumin, J).

To refine Polymerase I, Kornberg incubated strains of rapidly replicating E. coli bacteria with ATP and a substrate labeled with radioactive carbon (Kornberg, A). After many replications of the bacteria had occurred, Kornberg was able to isolate the enzyme using the radioactive labeling. When this enzyme was added to strands of DNA in the presence of the four nitrogenous bases, it caused the formation of additional, identical strands of DNA. Kornberg noted that for a reaction to occur in the presence of the enzymes, all four deoxynucleotides and DNA needed to be present. The product DNA accumulated until one of the substances was completed, resulting in around twenty more times the amount of DNA with which the experiment began. Each of the deoxynucleotides acted as limiting reactants. If one of the bases was not present, the DNA molecule was not built, and as soon as one base ran out, the DNA stopped replicating. Additionally, DNA needed to be present because DNA functions as a template in directing the synthesis of copies of itself. Without DNA, there was no template, and without all four bases present, synthesis stops with the unavailability of a bonding pair for one of the bases.

Arthur Kornberg knew a lot about enzymes, as he had studied them under the auspices of the NIH and Stanford University for many years. He identified the enzyme used above as unique (Hughes, S). In building the new DNA strand, it is taking direction from a preexisting template to add the specific purine or pyrimidine necessary to form the correct hydrogen bond with the base on the template. Kornberg provided five experimental bases for his hypothesis. (Kornberg, A)

First, Kornberg showed how the DNA molecule could only be replicated in the presence of heat, which would break the hydrogen bonds that hold together the double helix of the macromolecule. Once the molecule is split into two individual, pliable, strands, the enzymes attach nucleotide bases to the primer strands, resulting in two, identical, double-stranded macromolecules. No reaction occurred when the molecule was not heated, showing that the warmer environment was necessary for an enzymatic reaction to occur.

Second, Kornberg did experiments in which he replaced the purines and pyrimidines in the experiment with other molecules. These experiments showed that substitutions could be made in the bases only if there was no interference in the locations where hydrogen bonding needed to occur. For example, uracil was able to successfully replace thymine in an experiment, because the two molecules have locations for hydrogen bonding in the same places on their molecular surfaces. This experiment showed that it is not the deoxynucleotides that cause the reaction, as they can be replaced by similar molecules. Instead, an enzymatic reaction must be what attaches them to the template strand, as enzymes will cause a reaction with any molecule that will “fit” inside the enzyme.

Third, when the ratios of adenine to thymine and cytosine to guanine were identified, it was shown that enzymatically synthesized DNA has the same ratios as the natural, original DNA. Adding large amounts of any one of the bases did not change the ratio of the results. Thus, it is shown that the base completion was done through enzymatic synthesis with hydrogen bonding as the guiding mechanism, with large amounts of reactants not speeding up the reaction in any way.

Arthur Kornberg with his wife, Sylvy

Fourth, experiments were done to identify the sequences of nucleotides that occur by labeling the molecules with radioactive phosphate in each of the four bases four separate times. This experiment was done on DNA from many different sources. It was found that all sixteen possible sequences were apparent in each case, thought the pattern of frequency was unique to each source of DNA. The enzymatic replication showed a base-pairing of adenine to thymine and cytosine to guanine, and the replication produced two strands identical but facing opposite directions. This was the result accurately predicted by the Watson-Crick model.

Fifth, it was proven that DNA and all four of the bases needed to be present for replication to occur. Therefore, the origin of replication cannot be attributed to any one of them, and thus must be attributed to an external source: the synthesizing enzyme of DNA Polymerase I.

Arthur Kornberg and Severo Ochoa discovered the enzymes that opened the genetic code and provided the basis for genetic engineering. Their work on the synthesis of nucleotides and their assembly into DNA molecules has been the foundation of genetically designed chemotherapeutic agents currently used in the treatments of cancer, viral infections, and autoimmune diseases (Kornberg, A.). Their work has been the basis for diagnosis, treatment and prevention of disease. At no time in their research could these scientists have known how big a part their work would play in the development of biotechnology, yet their research has been the building blocks of some of the most revolutionary forms of medicine that are available today.

Works Cited

“Basic Research, the Lifeline of Medicine”. Nobelprize.org. Nobel Media AB 2014. Web. 5 Dec 2014.

Arthur Kornberg – Nobel Lecture: The Biologic Synthesis of Deoxyribonucleic Acid”. Nobelprize.org. Nobel Media AB 2014. Web. 5 Dec 2014.

Kornberg, in The Chemical Basis of Heredity, W. D. McElroy and B. Glass (Eds.),

Johns Hopkins Press, Baltimore, 1957, p. 579. Rev. Mod. Phys., 31 (1959)200.

Arthur Kornberg – Biographical”. Nobelprize.org. Nobel Media AB 2014. Web. 5 Dec 2014.

Hughes, Sally Smith. Interview with Arthur Kornberg: “Biochemistry at Stanford, Biotechnology at DNAX.” Regents University of California. 1988. Web. 5 Dec 2014.

Jochen Kumin. “Arthur Kornberg 1918-2007.” Access Excellence at the National Health Museum. 2009. Web. 5 Dec 2014.