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SUNY Downstate Sesquicentennial

Building a Legacy for the Future

Evelyn M. Witkin, PhD

Created the fields of DNA mutagenesis and DNA repair

Miriam H. Feuerman, PhD

photo

Evelyn Witkin is among the pioneers in the discovery of the role of mutation and repair in the function of the DNA in the genome. During her career, Evelyn answered every fundamental question about mutation induction by ultraviolet light. She discovered how mutations can be prevented by enzymatic repair of potentially mutagenic UV photoproducts in DNA by looking at curves on a plot. Her work laid the foundation for our understanding of genetic mutation and its contribution to human disease.

Chance played a recurring role in Evelyn's career just as it does in mutagenesis. She did her undergraduate work at New York University and received her PhD from Columbia University. She had intended to continue at NYU for her graduate studies and had earned a graduate assistantship. However, during her senior year in the fall of 1940, she learned that NYU was part of the gentlemen's agreement whereby African American athletes were left at home when the team traveled to schools in southern states. Incensed by this practice, she protested and as a result was suspended for three months along with the other student leaders of the protest. The suspension meant that Evelyn could not graduate with her class in May, could not start graduate school in the fall, and lost her graduate assistantship. That summer she obtained enough credits to graduate in October and was accepted to the Columbia zoology graduate program.

Evelyn was initially enchanted with the writings of Trofim Lysenko, the Russian plant breeder who didn't believe in the existence of genes but claimed that the environment could influence inherited characteristics. After further investigation, she learned that Lysenko's experiments and conclusions were flawed and even fraudulent. However, Evelyn's short infatuation with Lysenko was in large measure responsible for her desire to work with Professor Theodosius Dobzhansky at Columbia because of his ability to read Russian. Evelyn's interest in Lysenko's writings ended when she read papers by Hermann Muller. Muller wrote in 1922 that it was vital to explore how a gene could make copies of itself, how it could be changed, and how those changes could be propagated. His papers would influence her life's work.

When Evelyn started her graduate work in 1941, DNA, called a "dumb molecule" by Max Delbruck, was thought to consist only of a simple repeating sequence of the four nucleotides, not nearly complex enough to be the molecule of heredity. DNA was not known as the molecule of inheritance until the publication of Avery's work in 1944 and Hershey and Chase's work in 1952. [See Sternberg and Avery monograph. Ed.] The structure of DNA would not be reported until 1953, with its important implications for replication and repair. The nature of the major photoproduct induced by UV-light, pyrimidine dimers, would not be discovered until 1960.

Working under the supervision of Dobzhansky, Evelyn started inducing mutations in Drosophila using chemical agents. That changed when she read a preprint of the classic 1943 paper by Luria and Delbruck and learned that Escherichia coli had genes like other organisms and was an ideal system for studying genes and mutations. Dobzhansky arranged for Evelyn to work in bacteria with Dr. Milislav Demerec at the Carnegie Institution of Washington's Department of Genetics at Cold Spring Harbor (now known as Cold Spring Harbor Laboratory), where they were properly equipped. Cold Spring Harbor was the epicenter of a revolution in genetics that was taking place using E. coli and bacteriophage as model systems to study the nature of the gene.

In Evelyn's first experiment at Cold Spring Harbor, she exposed millions of bacteria to UV light and obtained only four surviving bacterial colonies. She decided to test those colonies to determine why they had survived a level of radiation exposure that killed the rest of the population. When Evelyn opened the incubator to discover that she had isolated mutants resistant to UV radiation, she was hooked. She completed her PhD in bacterial genetics and eventually become a staff member at the Cold Spring Harbor Laboratory. The colonies isolated had a mutation conferring a hundred-fold increase in resistance to both UV light and x-rays. This was the first demonstration that a single gene mutation could increase radiation resistance, an important discovery that became the subject of her doctoral dissertation.

Evelyn came to Downstate in 1955. Her husband, Dr. Herman Witkin, was a well-established member of the Downstate faculty. The commute from Cold Spring Harbor was getting more difficult, taking time away from more important things and, with a young family, neither parent liked being separated by that much distance. Evelyn secured a position at Columbia, where they planned to live, with Herman commuting to Brooklyn. The Witkins took a walk around the neighborhood surrounding Columbia and found it uninviting, so luckily for us, they changed their plans. Evelyn would find a position at Downstate and the family would move to Brooklyn. At that time, there were anti-nepotism laws preventing Downstate from paying both Evelyn and Herman but, fortunately, Evelyn could pay her own salary from a grant awarded by the National Institutes of Health. In the beginning, the only lab space available was part of a bench in Kings County Hospital in the lab of Dr. Robert Austrian in the Department of Medicine. [See Austrian monograph. Ed] Evelyn would work, mostly alone or with a lab technician, at the bench during the hours her children were in school and then work at home to analyze data and design more experiments. She started working this flexible schedule at Cold Spring Harbor with the advocacy of Vannevar Bush, whom Evelyn credits for progressive attitudes toward women in the work place.

Evelyn concluded in the early 1960s that an unrepaired UV photoproduct in DNA is lethal, because DNA polymerase, the normal DNA copying enzyme, stalled at these lesions. She proposed that the bacterium survives, albeit as a mutant, only if it has another DNA polymerase that is able to replicate past the lesion, even if there is a high probability of error in DNA synthesis. Not until 40 years later did biochemists isolate and purify the so-called error-prone DNA polymerase that is responsible for UV-induced mutations in E. coli. Human cells have many similar error-prone DNA polymerases. These enzymes are largely responsible for mutations caused by radiation and carcinogenic chemicals, and are now an important focus in cancer research.

As a member of the faculty of Downstate from 1955 to 1971, Evelyn worked mostly to elucidate mutation frequency decline or MFD. Starting with a strain requiring tryptophan for growth, and selecting UV-induced tryptophan-independent mutants, she had found that the mutation yield at any given UV dose was not determined at the instant of irradiation, but depended on post-irradiation metabolism. If the growth medium during the first ten minutes after exposure supports active protein synthesis, the induced mutation yield is very high, but if protein synthesis is inhibited during that time, over 90 percent of the induced mutations are irreversibly lost. That loss of induced mutations is MFD. By analyzing the kinetics of MFD under a variety of conditions, Evelyn was able to deduce almost all of the features of the mutagenic process. She concluded that MFD is due to the rapid enzymatic repair of the potentially mutagenic UV photoproducts when protein synthesis is inhibited or delayed. In the early 1960s, she wrote that MFD is repair before replication; mutation fixation is replication without repair.

Evelyn's work on MFD was the first to reveal the action of an anti-mutagenic repair process, later identified as excision repair, which is found in humans and most other organisms. She found that bacterial mutants lacking excision repair are exceedingly sensitive to radiation, and that the survivors of exposure are riddled with radiation-induced mutations. Human patients deficient in excision repair have xeroderma pigmentosum and suffer from extreme radiation sensitivity leading to multiple cancers, most commonly melanoma. In 1966, Evelyn isolated an MFD-deficient mutant and mapped the mutation to a gene locus on the E. coli chromosome she called mfd. Thirty-five years later, in 1991, the product of this gene was identified as an enzyme required for the rapid repair of actively transcribing genes. Humans deficient in this activity, transcription-coupled repair, are cancer-prone and may suffer from Bloom's syndrome, Cockayne's syndrome, or a form of progeria, a premature aging syndrome.

Toward the end of her stay at Downstate, Evelyn showed that UV damage of DNA leads to a number of effects in E. coli, including the induction of a latent bacteriophage and a delay in cell division leading to filamentous growth. She proposed that UV-damaged DNA generates a regulatory signal that activates a large number of genes. This was the first step toward her major discovery that became known as the SOS response. The term "SOS" was borrowed from the international danger signal because the SOS response in E. coli is activated only when the bacteria are under stress. In fact, when DNA is damaged to the point where it cannot replicate, more than 40 genes are activated, all contributing to the repair of the DNA and survival of the damaged cell. In a healthy cell, expression of the SOS genes is repressed. When the DNA is severely damaged, the SOS genes are coordinately de-repressed, and their products go into action until the damage is repaired. Only then does DNA replicate and the cell divide, and the expression of the SOS genes return to their normal state. One of the SOS genes in E. coli encodes the error-prone DNA polymerase that performs trans-lesion DNA synthesis and is responsible for UV photoproduct-induced mutagenesis. Evelyn continued to study the SOS response until her retirement in 1991.

Evelyn did most of her work before the molecular tools that are routinely used in labs today were even invented. Her experimental approach was to let the bacteria perform the relevant biochemistry, which is very different than what most biological investigators use today. She would perform very simple experiments with very intricate thinking behind them, using mainly Petri dishes, pipettes, and bacterial culture. The last laboratory step was counting the number of bacterial colonies found on plates. By the time she left Downstate in 1971, Evelyn achieved a detailed understanding of how UV photoproducts in DNA lead to mutations by interpreting curves on a graph. She showed that most UV-induced mutations are mistakes in replication caused by unrepaired photoproducts in DNA passing through the replication fork. Only very recently has this process been demonstrated using purified proteins in vitro.

Although Evelyn denies it, Louis Pasteur's remark applies to her: "Chance favors the prepared mind." In hindsight and with the benefit of subsequent molecular analysis, it is clear that Evelyn's experiments were successful because she consistently asked important questions and designed experiments that allowed her to deduce the correct answers from her data. Her work has been used as a guide for the biochemical and molecular characterization of DNA mutation and repair that was to follow. Remarkably, each conclusion she made was verified by others years and even decades later.

All the while that Evelyn was studying bacteria, she firmly believed she was studying cancer. This was never more evident than in 1983 when, as a member of the Douglass College at Rutgers University faculty, she read a paper published in the Proceedings of the National Academy of Sciences about p53 before its true role in cancer development was known. She read that p53 could cause a delay in the cell division, reminiscent of the filamentous growth response in bacteria after UV irradiation. She ran to her upstairs colleague, Warren Maltzman, practically with her UV light in hand, suggesting that he try exposing cells to UV light to see what happened to the levels of p53. A few days later, he excitedly reported to Evelyn that p53 levels were greatly increased in response to DNA damage caused by UV light or chemicals. He later published in Molecular and Cellular Biology that p53 levels increased in response to DNA damage through protein stabilization, a finding of considerable interest to cancer researchers.

This is an example of how Evelyn's work would have a role in our understanding of cancer and carcinogenesis, as she always believed it would. This entire line of research was spawned by what Evelyn calls "random collisions," which she believes can have an enormous positive impact on scientific progress. Her collegial attitude was likely developed during her training at Cold Spring Harbor. She was there in the 1940s, a golden age of research in bacterial and bacteriophage genetics. In addition to the guidance she received from Demerec, there were frequent "random collisions" with Salvador Luria, Max Delbruck, and Barbara McClintock.

Retirement has enabled Evelyn to return to some of her other interests. She has continued to follow advancements in biological science, and genetics in particular, with a broader point of view. The discovery that the human genome encoded fewer than 25,000 genes instead of the predicted 100,000 genes was the biggest surprise to her. She is less surprised by the discovery that nonprotein coding regions of the genome are transcriptionally active and may play a role in gene regulation. As a devotee of Darwin and the power of selection, she never really believed that the human genome could carry so much "clunky junky DNA going forever in a genome when it wasn't needed," she said. Evelyn is also a fan of Darwin's contemporary, the poet Robert Browning. She has uncovered evidence that Darwin and Browning were both influenced by the same seventeenth century book: The Wonders of the Little World, by Nathaniel Wanley. She became such a respected Browning scholar that she was elected vice president of the New York Browning Society.

Evelyn has been the recipient of many honors and awards. When she left Downstate in 1971 to join the faculty of Rutgers University, she was named the Barbara McClintock professor. In 1977, she was elected to the National Academy of Sciences. Very few women had come before her in that particular honor, so the diploma used only masculine pronouns. After some discussion with David R. Goddard, then the home secretary of the National Academy of Sciences, the diploma was rewritten to remove gender references. In 2000, she was awarded the Thomas Hunt Morgan medal by the Genetics Society of America. In 2002, Evelyn was awarded the President's national medal of science for "her insightful and pioneering investigations on the genetics of DNA mutagenesis and DNA repair that have increased our understanding of the processes as varied as evolution and the development of cancer." In 2004, she was awarded the distinguished research award by the New Jersey Association for Biomedical Research.

Acknowledgement

I would like to thank Evelyn Witkin for graciously agreeing to speak with me in the first place and for all her subsequent help with this piece.

References

  • Witkin, E. M. (2002) Chances and Choices: Cold Spring Harbor 1944-1955. Annual Review of Microbiology 56:1-15
  • Gross, C. (2001) The 2000 GSA Honors and Awards. The 2000 Thomas Hunt Morgan Medal Evelyn M. Witkin. Genetics 157:459-469
  • Witkin, E. M. (1989) Ultraviolet Mutagenesis and the SOS Response in Escherichia coli: A Personal Perspective. Environmental and Molecular Mutagenesis Supplement 14:30-34
  • (1969) Ultraviolet-Induced Mutation and DNA Repair. Annual Revue of Genetics 3:525-552
  • (1956) Time, Temperature, and Protein Synthesis: A Study of Ultraviolet-Induced Mutation in Bacteria. Cold Spring Harbor Symposia on Quantitative Biology 21:123-140