Astonishing Machines of Replication: Stanford, 1970-Present
By the early 1970s, ongoing discoveries (such as the repair ability of DNA polymerase and the existence of several other polymerases) made it clear to molecular biologists that the DNA synthesis and replication process was far more complex than anyone had imagined. Continuing their exploration of the synthesis process, Kornberg and his team pursued the question of how new chains of DNA are started. The 1967 synthesis work suggested that DNA polymerase could start a new chain, not just build a complete chain from a short length of primer DNA. Subsequent experiments had proved, however, that it could not.
One of Kornberg's former postdoctoral fellows, Reiji Okazaki, resolved one big question about how replication proceeds as the parental strands of the DNA helix come apart: The two strands run in opposite directions; one of the free ends, known as the 5´ end, has a free phosphate group on it; the other (the 3´ end) has a free -- OH group on it. (The 3 and 5 designations refer to the position of carbon atoms on the ring structure of the deoxyribose molecule.) DNA polymerase can assemble nucleotides continuously along the template strand only from the 5´ end. How then does the other strand function as a template? Okazaki found that copies of shorter segments of that 3´ strand were generated repeatedly, then joined by the ligase enzyme.
How were these shorter segments (known as Okazaki fragments) started? And what did the three known polymerases do, if anything? Kornberg turned again to the tiny bacterial viruses Phi X 174 and M13 to study the problem, and for the next two decades his team painstakingly elucidated the processes occurring at the replication fork, where the double helix came apart and duplication of each strand began. They identified a complex of seven different proteins that initiated the DNA chains and a multi-component enzyme, which they named DNA polymerase III holoenzyme, which completed the assembly of the new DNA. In subsequent work, they explored the chemical mechanism of the trigger that initiates chromosome replication during cell division.
Kornberg's work also provided the foundation for a concurrent development at Stanford during these years: recombinant DNA (rDNA), pioneered in part by Kornberg's colleague Paul Berg and Peter Lobban, then a graduate student in the department. As Kornberg later noted,
Significant roots of genetic engineering grew in the Biochemistry Department at Stanford because we had discovered or were applying the reagents basic to the manipulation of DNA: polymerase to synthesize long chains of DNA and fill in gaps, ligase to join contiguous ends of chains, exonuclease III to remove obstructive phosphate groups at chain ends, phage lambda exonuclease to chew back one end of a DNA chain, and terminal transferase, an enzyme . . . to add nucleotides, willy-nilly, to the other end of a DNA chain. These five enzymes were among the reagents that stimulated and nourished the two Stanford experiments that introduced recombinant DNA technology and led to the engineering of genes and chromosomes.
None of those hunting the enzymes that make and break DNA ever imagined that they would become the essential tools for this sweeping technical advance. Since the late 1970s, however, these techniques for splicing together the DNA of unrelated species have made it possible to use microorganisms to make insulin, human growth hormone, vaccines, and other products for treating and preventing disease.
Like many researchers in molecular biology and related fields, Kornberg had to address the opportunities and dilemmas presented by genetic engineering, especially the interaction between academic researchers, biotechnology companies, and the pharmaceutical industry. Acknowledging the role of the latter in bringing rDNA products to market, he continued to be concerned about the effects of industrial secrecy on scientific research. Would the free flow of scientific information be stifled to protect various company interests and profits? In the wake of the profitable patents on rDNA held by Stanley N. Cohen, Herbert Boyer, and Stanford University, Kornberg and others were approached by various biotech and pharmaceutical companies regarding new genetic engineering ventures--but they were repelled by the heavily commercial focus.
In 1980, however, Alex Zaffaroni approached Kornberg and several colleagues about founding a research institute to support the development of novel therapeutic products based on recombinant DNA technology. Zaffaroni, a biochemist by training, had started his career at Syntex Laboratories (which was the first to synthesize the hormone progesterone on an industrial scale) and risen to be president and CEO there. He later founded his own company, ALZA, to pursue development of innovative drug-delivery systems. Kornberg, impressed with Zaffaroni's integrity and commitment to the scientific side of the business, had served on ALZA's scientific advisory board since the late 1960s. Kornberg, Paul Berg, and biologist Charles Yanofsky became founding partners of the DNAX Institute of Molecular and Cellular Biology later that year. With their assistance, the institute was able to recruit some of the best young scientists. Their key policy was to provide the best resources in an open working atmosphere, allow the staff to publish freely, and thereby guarantee their scientific viability. This proved to be a wise strategy, and DNAX has been a successful enterprise. It was acquired by Schering-Plough Pharmaceuticals in 1982, and recently became part of the company's Biopharma research branch, where research is focused on immunological and oncological products. Kornberg continued to serve on the board of directors and assist with recruitment.
He also remained active in his laboratory research. In 1991, after many decades of work on DNA replication, he shifted his research focus to inorganic polyphosphate (poly P). He had been interested in this phosphate polymer since the 1950s, when he found an enzyme in E. coli that makes it. Poly P is found in many organisms but its functions were not well understood; many chemists regarded it as a "molecular fossil," perhaps once useful but now vestigial. Kornberg, however, found many likely functions for it, depending on its abundance, chain length, biologic source, and location in the cell. These include being an energy supply and ATP substitute, and a binder of metallic ions. Poly P also seems to help regulate responses to stresses and adjustments for survival in the stationary phase of cell growth and development, and plays a role in the motility and virulence of some disease-causing organisms. Kornberg continued the Poly P studies until several days before his death on October 26, 2007, at age 89.
Reflecting on his long scientific career, Kornberg has noted that although he has been a teacher, an administrator, and an author, "for me it was the research that mattered the most, because all of these other activities and the attitudes I brought to them were shaped by it."