[Editor’s note: While best known for his discovery of DNA polymerase and his subsequent synthesis of DNA molecules, Nobel Prize-winning biochemist Arthur Kornberg considers himself first and foremost a researcher of enzymes. Over the course of his career, which now spans a half-century, he has continued to pursue the study of these catalytics as other biochemist s—much to his chagrin—have chosen to ignore them. “Without knowing and respecting enzymes, better still loving them, answers to the most basic questions of growth, development, and disease will remain beyond reach,” he has said. Today, at the age of 71, Kornberg continues his enzyme research at Stanford University, where he has been a professor and chairman of the biochemistry department. He also continues to lecture; when contacted by The Scientist at Stanford !ast month, he was about to depart for Seoul, South Korea, where he was to make a presentation to the Congress of the Federation of Asian and Oceanian Biochemists. In the following reminiscence, Kornberg looks back on some of his earliest and most important years as a scientist and his multiple roles of discoverer, teacher, author, and administrator.]
Today, at the age of 71, Kornberg continues his enzyme research at Stanford University, where he has been a professor and chairman of the biochemistry department. He also continues to lecture; when contacted by The Scientist at Stanford !ast month, he was about to depart for Seoul, South Korea, where he was to make a presentation to the Congress of the Federation of Asian and Oceanian Biochemists.
In the following reminiscence, Kornberg looks back on some of his earliest and most important years as a scientist and his multiple roles of discoverer, teacher, author, and administrator.]
The current generation of scientists may be surprised to know that I had no formal research training. I was well started in a career of clinical medicine until World War II placed me in the National Institutes of Health (NIH), where I soon became an eager investigator of rat nutrition. Three years later, in 1945, I responded to the lure of enzymes and have remained faithful to them.
Science was unknown in my family and circle of friends. Once, in 1947, when I was in the biochemistry department of Washington University in St. Louis, working under the guidance of Carl and Gerty Con, Gerty told me that Carl had collected beetles and butterflies in his youth, and then asked: “Arthur, what did you collect?” “Matchbook covers,” was my sheepish response. What They were the dominant flora in the Brooklyn streets where I played and in the subways.
My early education in grade school and Abraham Lincoln High School in Brooldyn was distinguished only by “skipping” a few grades and finishing three years ahead of schedule. I recall nothing inspirational from teachers or courses except encouragement to get good marks.
I chose the cachet of City College in uptown Manhattan over nearby Brooklyn College. Competition among a large body of bright and Excerpted with permission from the Annual Review of Biochemistry, Vol. 58. © 1989 by Annual Reviews Inc.
Motivated students was fierce in all subjects. I carried over my high school interest in chemistry, but the prospects for employment in college teaching or industry were dismal. For lack of graduate studies or research laboratories at City College then, these possibilities barely existed. At age l9 in 1937, with a Bachelor of Science degree and no jobs to be had in the depths of the Great Depression, I welcomed the haven that medical school would provide for four more years.
Throughout college I worked evenings, weekends, and holidays as a salesman in men’s furnishing stores. This left little time for study or sleep and none for leisure. With these earnings, a New York State Regents Scholarship of $100 a year, no college tuition, and frugal living, I saved enough to see myself through the first half of medical school at the University of Rochester.
I enjoyed medical school and the training to become a doctor. Among my courses, biochemistry seemed rather dull. The descriptive emphasis on the constituents of tissues, blood, and urine reflected biochemistry in the United. States in the 1930s. The dynamism of cellular energy exchanges and macromolecules was still unknown, and the importance of enzymes had not penetrated my course or textbook. By contrast, anatomy and physiology presented integrated and awesome structures and functions. The aberrations presented in pathology and bacteriology were absorbing, as were the responsibilities to diagnose and treat patients during the clinical years.
Did I as a medical student consider a career in research? Not really. I expected to practice internal medicine, preferably in an academic setting; the idea of spending a significant fraction of my future days in the laboratory had no appeal. The medical school of the University of Rochester granted some students fellowships to take a year out for research. I had hoped but failed to get such an award from any of the departments. In those years, ethnic and religious barriers were formidable, even within the enlightened circle of academic science.
I did some research on my own, which grew out of curiosity about jaundice. I had noticed a slightly yellow discoloration of the whites of my eyes, and found that my blood bilirubin level was elevated and my tolerance to injected bilirubin reduced. I made similar measurements on as many medical students and patients as I could. I collected samples at odd moments and did the analyses on a borrowed bench, late at night and on weekends. The report I published called attention to the frequent occurrence of high bilirubin levels and reduced capacity to eliminate bilirubin, now recognized as signs of the benign familial trait called Gilbert’s disease.
Looking back, I realize that I enjoyed collecting data. I kept on collecting bilirubin measurements during my internship year and started setting up to 40 more analyses in the small sickbay of a Navy ship soon after I joined the service. The publication of my student work on jaundice attracted attention and led to my transfer from sea duty to do research at NIH, a rare assignment at that time. Discovery of DNA Polymerase (1955-1959).
Having learned how the likely nucleotide building blocks of nucleic acids are synthesized and activated in cells, it seemed natural that, in 1954, I would look for the enzymes that assemble them into RNA and DNA. Such an attempt might have been considered by some as audacious. Synthesis of starch and fat, once regarded as impossible outside the living cell, had been achieved with enzymes in the test tube. But the monotonous array of sugar units in starch or the acetic acid units in fat was a far cry from the assembly of DNA, thousands of times larger and genetically precise.
Yet, I was only following the classical biochemical traditions practiced by my teachers. It always seemedto me that a biochemist devoted to enzymes could, if persistent, reconstitute any metabolic event in the test tube as well as the cell does it. In fact better! Without the constraints under which an intact cell must operate, the biochemist can manipulate the concentrations of substrates and enzymes and arrange the medium around them to favor the reaction of his choice.
I have adhered to the rule that all chemical reactions in the cell proceed through the catalysis and control of enzymes. Once, in a seminar on the enzymes that degrade orotic acid, I realized that my audience in the Washington University chemistry department was drifting away. In a last-ditch attempt to gain their attention, I pronounced loudly that every chemical event in the cell depends on the action of an enzyme. At that point, Joseph Kennedy, the brilliant young chairman, awoke: “Do you mean to tell us that something as simple as the hydration of carbon dioxide [to form bicarbonate] needs an enzyme?” The Lord had delivered him into my hands. “Yes, Joe, cells have an enzyme, called carbonic anhydrase. It enhances the rate of that reaction more than a million-fold.” By 1954, the rapidly growing Escherichia coli cell had become a favored object of biochemical and genetic studies, and for me had replaced yeast and animal tissues as the preferred source of enzymes. To explore the synthesis of RNA, Uri Littauer, a postdoctoral fellow, and I prepared [14C-adenine]-ATP and maintained it as ATP with a regenerating system. Upon incubation with an E. coli extract, a small but significant amount of the radioactivity was incorporated into an acid-insoluble form, presumably RNA, and we proceeded eagerly to purify the activity responsible.
I also pursued the synthesis of DNA. Here, Ihad the invaluable help of Morris Friedkin, who had synthesized ‘4C-thymidine and was studying its uptake into the DNA of rabbit bone marrow or onion root tip cells. Disinclined to work with cell-free extracts, he generously saved the spent reaction fluid from which I recovered radioactive thymidine to use in trials with extracts of E. coli.
The results were mixed. Very little thymidine was incorporated into the acid-insoluble form indicative of DNA, only about 50 cpm out of the million with which we started. On the other hand, 5% to 10% of the thymidine was converted to novel soluble forms that resembled the phosphorylated states of the nucleo tide building blocks, possibly better than thymidine for DNA synthesis.
At this juncture, Herman Kaickar on a visit to St. Louis brought us the startling and unsettling news that [Severo] Ochoa and Marianne Grunberg-Manago, a postdoctoral fellow, had just discovered the enzymatic synthesis of RNA. It was for them a totally unexpected finding made while exploring aerobic phosphorylation in extracts of Azotobacter vinelandii. They observed an exchange of phosphate into ADPand the reversible conversion of ADP (or other nucleoside diphosphates) into RNA-like chains, and they named the enzyme polynucleotide phosphorylase.
On the strength of this new information, we shifted to using ADP rather than ATP in our studies with E. coli. The rate and extent of reaction were far greater, and we readily purified the enzyme involved. We had made a classic blunder. Accounting for a phenomenon does not insure that it is the only or the best explanation of it. In this instance, we were diverted from the discovery of RNApolymerase, which depends on ATP. By switching to ADP, we tracked the synthetic activity of polynucleotide phosphorylase and missed the key enzyme for gene transcription.
Ten months had passed before I repeated the experiment of converting radioactive thymidine to an acid-insoluble form. Once again, only a tiny amount of this presumed precursor was converted. But several things were different. For one, the radioactivity of the thymidine happened to he three times as great, and so the results seemed more impressive. For another, believing I had lost out on the synthesis of RNA, the synthesis of DNA became a more precious goal. Finally, I exposed the product to pancreatic DNase and found it became acid-soluble, a strong indication that it was DNA.
Even before I calculated the DNAse results, I stopped to tell Bob Lehman about them. Although his postdoctoral problem was well started, he was eager to switch to DNA synthesis. Progress was rapid. Bob soon found that thymidine phosphate was a far better precursor than thymidine and later showed that thymidine triphosphate was much better still. With improvements in the assay of DNA synthesis by these crude extracts, our goal was to purify the enzyme that assembled nucleotides into a DNA chain, the enzyme we would name DNA polymerase.
The most complex and revealing insights into the reaction would come from exploring the function of the DNA that I had included in the reaction mixture in my earliest attempt to incorporate thymidine into DNA. Some assume that DNA was included to serve as a template and that its primer role emerged many years later. Not so. I added DNA expecting that it would serve as a primer for growth of a DNA chain, because I was influenced by the Con work on the growth of carbohydrate chains by glycogen phosphorylase. I never thought that I would discover a phenomenon utterly unprecedented in biochemistry: an absolute dependence of an enzyme for instruction by its substrate serving as a template.
I had added DNA for another reason. Nuclease action in the extracts was rampant, and I wanted a pool of DNA to surround the newly incorporated thymidine and protect at least some of it. Only later did Lehman and I learn that the added DNA fulfilled two other essential roles. It indeed served as a template and also as a source of the missing nucleotides. The DNA was cleaved by DNases in the extract to nucleotides. These were converted by ATP and five kinases in the extract to the diand triphosphates of the A, G, C, and T deoxyribonucleotides, which were then still unknown.
Creation of Life in the Test Tube (1960-1967)
With purified DNA polymerase, we could show that the DNA product reflected the base composition of the template and the frequencies of the 16 possible dinucleotides. The “nearest neighbor” sequence method, which we devised to determine the dinucleotide frequencies, also revealed that the two strands of the double helix have opposite ‘polarities, a structural feature that had not been experimentally demonstrated up to that time.
We also made the unexpected discovery that the enzyme, in the apparent absence of any template would, after a considerable delay, make DNA-like polymers of simple composition: the alternating copolymers polydA*dT and polydG*dC and the homopolymer pairs of polydA with polydT and of polydG with polydC. These polymers, once made, proved to be superior templates and have been widely used in DNA chemistry and biology. Generation of the polymers de novo could be ascribed to the reiterative replication of short sequences in the immeasurably small amounts of DNA that contaminate a polymerase preparation.
For more than 10 years, I had to find excuses at the end of every seminar to explain why the DNA product had no biologic activity. If the template had been copied accurately, why were we unsuccessful in all our attempts to multiply the transforming factor activity of DNA from Pneumococcus, Hemophilus, and Bacillus species? Finally, with the arrival of ligase in 1967, a crucial test could be made. Mehran Goulian and I could replicate the single-stranded circle of phage FIX ITTI 174 with DNA polymerase and then seal the complementary product with ligase. The circular product was isolated and then replicated to produce a circular copy of the original viral strand, which could be assayed for infectivity in E. coli. We found the completely synthetic viral strand to be as infectious as that of the phage DNA with which we started!
After so many years of trying, we had finally done it. We had gotten DNA polymerase to assemble a 5000-nucleotide DNA chain with the identical form, composition, and genetic activity of DNA from a natural virus. All the enzyme needed was the four common building blocks: A, G, T, and C. At that moment, it seemed there were no major impediments to the synthesis of DNA, genes, and chromosomes. The way was open to create novel DNA and genes by manipulating the building blocks and their templates.
In a very small way, we were observers of something akin to what those at Alamogordo on a July day in 1945 witnessed in the explosive force of the atomic nucleus. Harnessing the enzymatic powers of the cellular nucleus had neither the dramatic staging of light and sound nor the stunningly apparent global consequences. Yet, this demonstration of our power with enzymes that build and link DNA chains would soon help others forge a different revolution, the engineering of genes and modification of species.
Scientist, Teacher, Author, Chair- man: In What Order?
In May 1988, when my former and present students and colleagues gathered in San Francisco for a gala 70th birthday party, I thought it would be fun to select from the 30 or more enzymes I had worked with, the 10 that I favored most. I was surprised to find that six of the 10 were discovered in the brief period from 1948 to 1955: nucleotide pyrophosphatase, NAD synthetase, phoshatidic acid synthetase, PRPP synthetase, polyphosphate synthetase, and DNA polymerase. That left only four to be selected from more than 20 enzymes that appeared in the next 30-plus years. In as much as some of the enzymes omitted from the top 10 are far more deserving of selection than some of the chosen ones, it is clear that the basis for the choice of the first six was largely sentimental. I was most attached to those enzymes that came during the time when I collected the data myself, from conception to delivery.
In my marriage to enzymes, I have found a level of complexity that suits me. I feel ill at ease grappling with the operations of a cell, let alone those of a multicellular creature. I also feel inadequate in probing the fine chemistry of small molecules. Becoming familiar with the personality of an enzyme performing in a major synthetic pathway is just right. To gain this intimacy, the enzyme must first be purified, and I have never felt unrewarded for any effort expended this way. I once shocked the dean of the Washington University School of Medicine by telling him that my prime interest as chairman of the Department of Microbiology was to do and foster research rather than teach. It has never been otherwise. Experiments are far more consuming and fulfilling for me than any form of teaching. Still, I have enjoyed a rather modest amount of formal lecture and laboratory instruction and have done it conscientiously. For the student, didactic teaching fails without the infusion of scientific skepticism and a fervor for new knowledge, and these things are naturally conveyed by someone dedicated to research.
The most rewarding teaching for me has been in the intimate, daily contact with graduate and postdoc toral students. Well over a hundred of them spent from two to five years in my laboratory and were exposed to my tastes and my obsession with the use of time. I felt closest to those who shared my devotion to enzymes and my concern with the productive use of our most precious resource: each of the hours and days that so quickly stretch into the few years of a creative life.
Imagination or hard work? At either extreme—speculating about complex phenomena or doggedly collecting data—success may come on occasion and draw some acclaim. But the most consistent approach for acquuing a biochemical understanding of nature lies in between. The novel is yet to be written that captures the creative and artistic essence of ‘scientific discoveries and dispels images of the scientist as dreamer, walking in the woods awaiting a flash of insight or of the scientist as engineer, at an instrument panel executing a precisely plarmed experiment. Some intermediate ground, hard work with a touch of fantasy, is what I have sought for myself and my students.
If asked to name varieties of mental torture, most scientists would place writing near the top of the list. As a result, scientific papers are usually put off or dashed off and demean the quality and value of the work they describe. Writing a paper is an integral part of the research and surely deserves the small fraction, say 5%, of the time spent finding the thing worth reporting. Yet, I feel uneasy seeing students and colleagues writing at their desks during “working hours” rather than busy at the laboratory bench Whereas taking time to prepare a scientific report is unavoidable, writing a book always seemed an unconscionable abdication from research until I wrote one.
Writing DNA Synthesis (San Francisco, W.H. Freeman, 1974), a 400-page book, was a surprise in many ways. First, the effort was far greater than I had imagined. Very little from lecture notes and reprints could be lifted and placed in the right context and still remain readable. I was also surprised by the pleasure I found in reworking and polishing sentences and paragraphs for brevity and clarity, a satisfaction I had never found in crossword puzzles.
Having regarded teaching and book writing as deviant activities for a dedicated scientist, then surely the administrative work of a departmental chairman should be beyond the pale. Yet, I served as chairman for more than 20 years and never found it a serious intrusion on my time or attention. On the contrary, the benefits of creating and maintaining a collegial and stimulating scientific circle were well worth the investment I made.
Involvement in medical school and university affairs is a far different matter. I never found the skills and patience to function at these levels. For me, the most burdensome feature of being a departmental chairman was the obligated service on the Executive Committee of the Medical School, preoccupied with budgets, promotions, interdepart mental feuds, and salaries. In six years at Washington University and 10 at Stanford, I cannot recall a deliberate discussion of science or educational policy.
Increasingly conspicuous in current scientific life are the extramural administrative and educational activities, which, with the attendant travel, may consume half the time of prominent members of a science faculty. I have done less than most, but have been unable to resist participating, particularly in writing essays, testifying for federal support of research and training, and most recently in the founding and development of a biotechnology enterprise [the DNAX Research Institute, Inc., later acquired by the Schering-Plough Corp.] with the mission of applying the techniques of molecular and cellular biology to the therapy of diseases of the immune system.
All these nonresearch activities, in and out of the university, fail to. give me a deep sense of personal achievement. In research, it is up to me to select a corner of the giant jigsaw puzzle of nature and then find and fit a missing piece. When after false starts and fumbling, a piece falls into place and provides clues for more, I take pleasure in having done something creative. By contrast, in my other activities, which are just as personal, all I do, it seems, is try to behave in a common sensical, fair, and responsible way, as anyone else would. With research so dominant over my teaching, writing, and administrative activities, in sharply descending order or importance to me, I sometimes wonder whether, valued for their contributions to science, this order might be inverted.
As for writing, the monographs on DNA replication, with more than 40,000 copies sold, have made it easier for others to enter and work in this field. More than offering a readable account of a forbiddingly specialized area of biochemistry, these books have helped revive an appreciation that enzymology provides a direct route toward solving biologic problems and creates reagents for the analysis and synthesis of a great variety of compounds for all branches of biologic science.
With regard to teaching, assumption of credit for the success of a student has always puzzled me. There simply are no controls in these experiments. How do I know, given a motivated, gifted student, whether I have been a help or hindrance? Nevertheless, having involved myself in the daily scientific lives of my students, I may have guided some of them in directions that attract me and thereby diverted them from a career in biology or chemistry to the love and pursuit of biochemistry and enzymes.
Finally, even were I forced to agree that my activities in administration, writing, and teaching had a singular quality, I would have to concede further that my discoveries in science did not. Very likely, they would have been made by others soon after. Yet in the last analysis, I will argue that for me it was the research that mattered most, because all my attitudes and activities were shaped by it.