Arthur Kornberg’s discovery that a fully infective viral DNA could be re-created in a test tube demonstrated that a purified enzyme could produce virtually error-free synthesis of a simple chromosome outside the living cell. It represented a major step forward in genetic science and engineering.
Until the mid-1940’s, it was believed that proteins were the carriers of genetic information, the source of heredity. Proteins, which are composed of many different amino acid units, appeared to be the only biological molecules that had the complexity necessary to encode the enormous amount of genetic information required to reproduce even the simplest organism. Nevertheless, proteins could not be shown to have genetic properties, and by 1944, it was demonstrated conclusively that deoxyribonucleic acid (DNA) is the material that can transmit hereditary information. DNA isolated from a strain of infective bacteria that cause pneumonia was able to transform a strain of noninfective bacteria into an infective strain, and the infectivity trait was transmitted to future generations. Subsequently, it was established that DNA is the genetic material in virtually all forms of life, except some viruses in which the genome is based on ribonucleic acid (RNA).
Once DNA was known to be the transmitter of genetic information, scientists sought to discover how it performs its role. DNA is a polymeric molecule composed of four different units, called deoxynucleotides. The units consist of a sugar, a phosphate group, and a base; they differ only in the nature of the base, which is always one of four related compounds: adenine, guanine, cytosine, or thymine. The means by which such a polymer could transmit genetic information was hard to discern. In 1953, James D. Watson
From their analysis of the structure of DNA, Watson and Crick inferred its mechanism of replication. Their work led to an understanding of gene function in molecular terms. Watson and Crick showed that DNA has a very long double-stranded (duplex) helical structure. In the duplex, the sugar and phosphate units of each deoxynucleotide are joined to form the exterior of the helix, and the bases form the interior. They proposed that the bases are arranged in a defined sequence, analogous to the arrangement of letters of the alphabet into words. The genetic code, the hereditary message, lies in the precise sequence of the bases.
DNA has a duplex structure, because each base forms a link to a specific base on the opposite strand. The linkage (or pairing) is most specific; adenine is always paired with thymine, and guanine with cytosine. The discovery of this complementary pairing of bases provided a model to explain the two essential functions of a hereditary molecule: It must preserve the genetic code from one generation to another, and it must direct the development of the cell. Watson and Crick proposed that DNA is able to serve as a mold (or template) for its own reproduction because the two strands of DNA polymer can separate. Upon separation, each strand acts as a template for the formation of a new complementary strand. An adenine base in the existing strand gives rise to the complementary thymine base in the new strand, guanine gives rise to cytosine, and so on. In this manner, a new double-stranded DNA is generated that is identical to the parent DNA.
Watson and Crick’s theory provided a valuable model for the reproduction of DNA, but it did not explain the biological mechanism by which the process occurs. The biochemical pathway of DNA reproduction and the role of the enzymes required for catalyzing reproduction were discovered by Arthur Kornberg and his colleagues. For his success in achieving DNA synthesis in a test tube and for discovering and isolating an enzyme—DNA polymerase—that catalyzed DNA synthesis, Kornberg was a corecipient of the 1959 Nobel Prize in Physiology or Medicine
To achieve DNA replication in a test tube, Kornberg found that a small amount of preformed DNA must be present, in addition to DNA polymerase enzyme and all four of the deoxynucleotides that occur in DNA. Although the polymerase enzyme was isolated from a strain of coli bacteria called Escherichia coli (E. coli), the preformed DNA did not have to come from E. coli. Preformed DNA isolated from almost any source—from different strains of bacteria, from a virus, or from calf thymus glands—served as a template with the E. coli DNA polymerase enzyme.
DNAs isolated from different sources have different base compositions—that is, different proportions of the base pairs adenine plus thymine and guanine plus cytosine. Kornberg discovered that the base composition of the newly made DNA was determined solely by the base composition of the preformed DNA that had been used as a template in the test-tube synthesis. This result showed that DNA polymerase obeys instructions dictated by the template DNA. It is thus said to be template-directed. DNA polymerase was the first template-directed enzyme discovered.
Although test-tube synthesis was a most significant achievement, important questions about the precise character of the newly made DNA were unanswered. Methods of analyzing the order, or sequence, of the bases in DNA were not available, and hence, it could not be shown directly whether DNA made in the test tube is a very accurate, exact copy of the template of DNA or merely an approximate copy. In addition, some DNAs prepared by DNA polymerase appeared to be branched structures. Given that chromosomes in living cells contain long, linear, unbranched strands of DNA, this branching might indicate that DNA synthesized in a test tube was not equivalent to DNA synthesized in the living cell. (Conversely, branching could be a technical artifact.) In Kornberg’s early experiments, he was unable to show whether the DNA synthesized was a new molecule or merely an extension of the preformed DNA. The technical problems were formidable, and the precision with which polymerase enzyme copied DNA chains, consisting of thousands of nucleotides, was unproved.
Kornberg realized that the best way to demonstrate that newly synthesized DNA is an exact copy of the original was to test the new DNA for biological activity in a suitable system. His first attempts to produce biologically active DNA, using bacterial DNA as a template, were unsuccessful. Failure, although disappointing, was not too discouraging because the lack of success could be attributed to technical problems in obtaining an intact, unfragmented DNA for the template. It is almost impossible to isolate an entire bacterial DNA molecule that is extremely long—4 million base pairs in length—without random fragmentation of the DNA and resultant loss in biological activity. In order to avoid the problem of fragmentation, the synthesis of a simpler DNA, a viral genome, was then attempted.
Robert L. Sinsheimer’s group of researchers were experts in the biology and biochemistry of a special bacterial virus that promised to be a good candidate for test-tube DNA synthesis. This virus has a circular, single-stranded DNA molecule approximately fifty-five hundred deoxynucleotides long, which folds upon itself and can be isolated intact. In nature, the virus reproduces inside an E. coli bacterial cell by producing double-stranded DNA as a replication intermediate. Identical copies of the original single-stranded viral DNA are made from the intermediate inside the cell to produce a new, infective virus. Sinsheimer and colleagues developed tests of viral infectivity that revealed that a change in even one nucleotide among the fifty-five hundred total in the viral DNA could make the virus noninfective.
Kornberg reasoned that a demonstration of infectivity in vital DNA produced in a test tube would prove that polymerase-catalyzed synthesis was virtually error-free and equivalent to natural, biological synthesis. The experiment, carried out between August and September, 1967, by Kornberg, colleague Mehran Goulian at Stanford University, and Sinsheimer at the California Institute of Technology, was a complete success. The viral DNAs produced in a test tube by DNA polymerase enzyme, using viral DNA template, were fully infective. This synthesis showed that DNA polymerase could copy not merely a single gene but an entire chromosome (containing eleven genes) of a small virus without error.
The purification of DNA polymerase and the preparation of biologically active DNA were major discoveries that influenced biological research on DNA for decades. Kornberg’s methodology proved invaluable in the discovery of other enzymes that synthesize DNA. These enzymes have been isolated from E. coli bacteria and from other bacteria, viruses, and higher organisms.
In the early 1950’s, when Kornberg first attempted to reproduce DNA, many scientists doubted that complex biological molecules could be reconstructed successfully outside the living cell. Although much was known about the degradation of complex molecules, little was known of their synthesis. A number of biochemists thought that the synthesis and breakdown of large biomolecules were two-way processes, proceeding by the same reversible pathways. Kornberg recognized that the biochemical route by which DNA is made is separate and distinct from the route by which it is degraded in the cell. His use of E. coli bacteria (after initial unsuccessful experiments with animal cells) was also an inspired choice. Kornberg reasoned that E. coli cell extracts should be a good source of DNA polymerizing enzymes because E. coli cells duplicate, and hence reproduce their DNA, very frequently (every twenty minutes). Although this choice was correct, the enzyme was difficult to isolate. For example, to obtain half a gram of DNA polymerase, 100 kilograms of E. coli cells had to be processed.
The test-tube preparation of viral DNA also had significance in the studies of genes and chromosomes. In the mid-1960’s, it had not been established that a chromosome contains a continuous strand of DNA. Kornberg and Sinsheimer’s synthesis of a viral chromosome proved that it was, indeed, a very long strand of uninterrupted DNA.
Experiments with E. coli bacterial mutants in the mid-1970’s led to an unexpected finding concerning the role of polymerase enzyme in DNA synthesis. Mutant E. coli bacteria were isolated which reproduced at normal rates, although the activity of their DNA polymerase enzyme was much diminished, reduced to 1 to 2 percent of normal. The apparent inconsistency between growth rates and polymerase activity was explained by the discovery that more than one enzyme is required to copy DNA in nature. DNA polymerase (now called DNA polymerase I) is not the main enzyme that reproduces DNA in the cell. Its main role is to “proofread” DNA, removing unwanted bases from growing DNA strands, and to repair damaged DNA. These findings revealed an important principle in biology: A function in nature cannot be ascribed to an enzyme on the basis of test-tube experiments alone; the use of mutants and the study of reactions inside the cell are also necessary.
Kornberg and Sinsheimer’s work laid the foundation for subsequent recombinant DNA research and for genetic engineering technology. This technology promises to revolutionize both medicine and agriculture. The enhancement of food production and the generation of new drugs and therapies are only a few of the benefits that may be expected to accrue to humankind.
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Berg, Jeremy M., John L. Tymoczko, and Lubert Stryer. Biochemistry. 6th ed. New York: W. H. Freeman, 2007. Part 1, “Molecular Design of Life,” and part 5, “Genetic Information: Storage, Transmission, and Expression,” provide excellent, current information on hereditary mechanisms and the means by which genetic information is expressed inside the cell. Chapters 4 and 26 describe concisely the structure of DNA and its mode of replication. Text is geared toward a student of science who has some knowledge of biochemical terminology. Includes useful references. -
Friedberg, Errol C. DNA Repair. New York: W. H. Freeman, 1985. This textbook is a useful complement to Kornberg’s DNA Replication (cited below). A valuable source for the research scientist and advanced student, the text provides current information on DNA damage, the effects of DNA damage, and the mechanisms by which an organism can repair its damaged DNA. -
Goulian, Mehran, Arthur Kornberg, and Robert L. Sinsheimer. “Enzymatic Synthesis of DNA, XXIV: Synthesis of Infectious Phage ϕX 174 DNA.” Proceedings of National Academy of Sciences 58 (1967): 2321-2328. This research article is the primary source for the reader seeking information on details of the means by which Kornberg and colleagues first synthesized biologically active DNA. -
Kornberg, Arthur. DNA Replication. San Francisco: W. H. Freeman, 1980. This outstanding and highly readable textbook is the prime resource for the research scientist or student interested in the biochemistry of DNA replication and metabolism. DNA replication in prokaryotes (bacteria and viruses) is covered most comprehensively; replication in eukaryotes (higher organisms) is also treated. Although some information is out of date, this text is still an invaluable source. -
_______. 1982 Supplement to DNA Replication. San Francisco: W. H. Freeman, 1982. Should be used in conjunction with Kornberg’s DNA Replication (cited above). Covers the extensive new discoveries made between 1980 and 1982. -
_______. “The Synthesis of DNA.” Scientific American 219 (April, 1968): 64-78. This article is addressed primarily to the nonspecialist who is interested in science. Provides a concise, popularized account of DNA synthesis. Though some information is out of date, this article is still essentially correct and has not been superseded. -
Zubay, Geoffrey L. Biochemistry. 4th ed. Dubuque, Iowa: Wm. C. Brown, 1998. A useful textbook for the advanced student. Chapter 26, “DNA Metabolism,” provides current biochemical information on DNA synthesis and breakdown. Other chapters in section 5, “Nucleic Acid and Protein Metabolism,” cover related biochemical processes.
Avery, MacLeod, and McCarty Determine That DNA Carries Hereditary Information
Bevis Describes Amniocentesis as a Method to Check Fetal Genetic Traits
Watson and Crick Announce the Double-Helix Model for DNA
Ochoa Creates Synthetic RNA
Horsfall Detects the Link Between Cancer and Altered DNA
Nirenberg Cracks the Genetic Code