DNA mutagenesis and DNA repair
Larry H. Bernstein, MD, FCAP, Curator
Leaders in Pharmaceutical Intelligence – Series E (2; 2.11)
Evelyn M. Witkin, born Evelyn Maisel is an American geneticist who was awarded the National Medal of Science for her work on DNA mutagenesis and DNA repair.
She earned her Ph.D. in 1947 with Theodosius Dobzhansky at Columbia University for her Drosophila research. Her interests evolved from Drosophila genetics to bacterial genetics, and she spent the summer of 1944 at Cold Spring Harbor, where she isolated a radiation-resistant mutant of E. coli. Witkin remained at the Carnegie Institution Department of Genetics at Cold Spring Harbor until 1955.
In 1971, she was appointed Professor of Biological Sciences at Douglass College, Rutgers University, and was named Barbara McClintock Professor of Genetics in 1979. Witkin moved to the Wakeman Institute at Rutgers University in 1983. Among her many honors are membership in the National Academy of Sciences (1977), Fellow of the American Association for the Advancement of Science (1980), American Women of Science Award for Outstanding Research, and Fellow of the American Academy of Microbiology. She was largely responsible for creating the field of DNA mutagenesis and DNA repair, which has played an important role in the biochemical sciences and in clinical radiation therapy for cancer. She is a member of the National Academy of Sciences, a Fellow of the American Association for the Advancement of Science, and the American Academy of Arts and Sciences. Dr. Witkin has also received the Thomas Hunt Morgan Medal of the Genetics Society of America in 2000 and the the Wiley Prize.
Oral History
The reason that I got into genetics in the first place, this is while I was an undergraduate [was that] a friend of mine, my first boyfriend, was a Harvard student who was quite a radical and he had gotten hold of [Trofim Denisovich] Lysenko’s papers. And we would read them and you know, Lysenko didn’t believe in the gene. He thought Mendel was a bourgeois nothing and didn’t believe in the gene and all you had to do was manipulate the environment and you could produce anything. And he was in power. You know, to Stalin this sounded great. You know the story. But at any rate, I didn’t know any genetics yet. So this sounded very nice. It would be wonderful to show that you could just manipulate the environment and change things. And I got intrigued and I thought this would be something to look into and my idea was in going into genetics was to test this! Actually it took about a month of genetics at Columbia to realize that he [Lysenko] was completely a fraud, but I never told Dobzhansky until his fiftieth [birthday] party. It was why I got into genetics and why I wanted a Russian advisor! I confessed, and that’s why—he thought it was absolutely hilarious.
When the Demerec lab was built we all had the opportunity to design our own laboratories. That was fun! And mine was right next to Hershey’s and that was a pleasure.
Well, he was an interesting man too. He was rather—you’ve probably heard from others—he was rather reserved and very focused on his work and not very sociable, but extremely generous and kind. And obviously very brilliant! He was very helpful to me.
I could tell you one story about the 1946 symposium, which I don’t know whether you’ve heard. It’s a story about [Salvador] Luria. He was one of the organizers of the program. That was the first symposium after the war and it was the first one on microorganisms and he and some others were getting the program together and he had seen a paper by a woman named Mary Bunting which was published in 1938. [She] worked with bacteria. He was very impressed by that paper. He said that was really the only paper he knows of that does real bacterial genetics before his work with Delbrück. And he said, “She has to be on the program. We have to find her and get her to come to the symposium.” Nobody knew where Mary Bunting was and he did some real hunting. He got other people to work on it, and I remember he was saying at one point, “We can’t have the symposium without her. We have to find her!” Well, they did find her. Her husband was on the faculty at Yale Medical School and they located her there. And she had three young children and was expecting a fourth. That was her Ph.D. Thesis—this paper [that Luria spoke about] She hadn’t been doing any scientific work for awhile and when Luria proposed that she come talk about this work at the symposium she was horrified and said she couldn’t possibly, and doesn’t even remember and “Go away!” And he was very insistent and she did turn up at the symposium and gave this wonderful talk. And it really changed her life forever. I don’t know if you [know], [but] she became the Dean of Radcliffe College.
But what happened at the symposium was, since she was bogged down with babies at Yale, some of the people here who were at Yale—[such as] Ed Tatum, I remember—they arranged for her to have some lab space at Yale and invited her to come and do some work whenever she had time. She came to seminars and caught up with things. And then she got a job when her children were a little bigger. I think her husband died around there too. She became the Dean of the Douglas College at Rutgers and she had done some very good work there in microbiology. And then she went on to become the Dean of Radcliffe and started the Mary Bunting Institute there, which is an institute designed to make it possible for women to resume their scientific careers after they’ve had a break for family reasons. She told me—I called her at one point to verify this because I mentioned this at some meeting, and I think I talked about it at the phage meeting in ’95—and she verified the fact and she said she would never have gone back to work if not for Luria’s having dug her out. Yes, it really shows things about Luria that I’m not sure are known. One is that he’s—he takes it for granted that men and women are equal. It didn’t occur to him that there’s a reason why she shouldn’t be there. And he also is very generous scientifically. He wanted her to have the credit for doing the first real genetics in bacteria.
Well, I guess I met Barbara [McClintock] my very first day here. I stayed at the dormitory and she was living there at the time. I guess I met here in the living room of the dormitory the very first day that I came and we started talking. I found her absolutely fascinating; she told me quite a lot about what she was doing. We became really good friends. And I spent a lot of time visiting her lab, and she developed a habit of calling me whenever she had something especially exciting. These were the years when she was beginning to discover transposition. I would just sort of drop everything and run over and she would show me something that was beginning to make sense to her and it was just such a privilege to be in that relation[ship] with her—to watch this story develop. It was unmistakably convincing as you explained it. You know, not having known very much about maize genetics it wasn’t easy for me to follow. But she was very patient about describing the experiments and she really was very confident about what she was doing.
Harvard geneticist wins Lasker Award for DNA work
Stephen J. Elledge Ph.D. is a Professor of Genetics at the Harvard Medical School. He earned his B.Sc. in chemistry from the University of Illinois at Urbana–Champaign and his Ph.D. in biology from MIT.
Education: Massachusetts Institute of Technology
Awards: Gairdner Foundation International Award, NAS Award in Molecular Biology, Lasker Award 2015
Harvard geneticist Stephen Elledge started his scientific career trying to figure out how to tinker with the DNA of human cells. Instead, he ended up eavesdropping on the process cells use to fix genetic mistakes.
This accidental work led to profound insights into DNA repair relevant to human birth defects, cancer, and aging.
Stephen J. Elledge, the Gregor Mendel Professor of Genetics and of Medicine at Harvard Medical School and Brigham and Women’s Hospital, is a co-recipient, with Evelyn Witkin of Rutgers University, of the 2015 Albert Lasker Basic Medical Research Award. The award, widely considered to be among the most respected in biomedicine, will be presented on Sept. 18 in New York City.
Elledge and Witkin are being honored for their seminal discoveries that have illuminated the DNA damage response, a cellular pathway that senses when DNA is altered and sets in a motion a series of responses to protect the cell. This pathway is critical to a better understanding of many diseases and conditions, such as cancer.
As cells divide and reproduce, they have to make precise copies of their DNA. Typos can doom a fetus, lead to birth defects, and cause cancer as well as symptoms of aging.
Elledge, who is also affiliated with Brigham and Women’s Hospital, uncovered a sequence of events, called a pathway, that protect a cell once its DNA has been damaged or incorrectly copied.
Harvard Medical School and Brigham and Women’s Hospital scientist recognized for discovering DNA repair
“Steve is an amazing scientist, mentor, and colleague,” said Jeffrey S. Flier, dean of Harvard Medical School (HMS). “His insights into the basic mechanisms of the DNA damage response have profoundly enriched our understanding not only of the fundamental genetics of all cellular life, but also of how we conceptualize many diseases and conditions, especially cancer. This distinction is richly deserved, and I am delighted that Steve is being honored for this extraordinary body of work.”
Stephen J. Elledge: Driven by Questions
Harvard geneticist Stephen J. Elledge has been recognized with the Lasker Award for a discovery that illuminates the DNA damage response. This pathway is critical to a better understanding of many diseases and conditions, such as cancer. Co-produced by Harvard Medical School and Brigham and Women’s Hospital.
“We are extremely proud of Steve, who is truly deserving of this recognition,” said Elizabeth G. Nabel, president of Brigham and Women’s Health Care. “Courageous and insatiably inquisitive, he represents the best of Brigham and Women’s and our mission of driving innovation in basic science to improve human health. As a devoted mentor, Steve is deeply committed to guiding the careers of young investigators, ensuring that the next generation of scientists is filled with curious, passionate, and talented researchers.”
Elledge often describes the process by which a cell duplicates itself as akin to the duplication of a small city. It is a vastly complex process that requires many levels of intricate coordination. Each cell contains a detailed blueprint for this entire process: DNA.
But not every duplication results in a perfect copy. That is because each time a cell makes a copy of itself, DNA is vulnerable to damage, not only from faulty cellular processes, but also from such entities as environmental chemicals. As DNA damage accumulates, it profoundly complicates a cell’s ability to make a faithful copy of itself. This can lead to serious illnesses, birth defects, cancer, and other health problems.
Witkin discovered how bacteria respond to DNA damage, detailing the response to UV radiation. Elledge uncovered a DNA-damage-response pathway that operates in more complex organisms, including humans.
Over the years, Elledge and his colleagues elucidated a signaling network that informs a cell when DNA sustains an injury. Called the DNA damage response, this network senses the problem and sends a signal to the rest of the cell so it can properly repair itself; otherwise, severe mutations could occur. As a result, this pathway helps keep the genome stable and suppresses adverse events such as tumor development. When individuals are born with mutations in this pathway, they often have severe developmental defects. If the pathway is interfered with later in life, cancer can result.
In addition to the award in basic medical research, the Lasker Foundation is also presenting awards to individuals in clinical research and in public service.
According to Claire Pomeroy, president of the Albert and Mary Lasker Foundation, this year’s recipients “remind us all that investing in biological sciences and medical research is crucial for our future.”
Joseph L. Goldstein of the University of Texas Southwestern Medical Center and chair of the Lasker Medical Research Awards Jury, added, “The 2015 Lasker winners had bold ideas and pursued novel questions that they tested through fearless experimentation.”
Over the past century, researchers have invested substantial efforts toward understanding the cell cycle. However, only recently have these studies gained a molecular foothold. Leading the research in this field is Stephen J. Elledge, professor of genetics at Harvard Medical School and Brigham and Women’s Hospital in Boston. Playing the dual roles of inventor and investigator, Elledge developed original techniques to define what drives the cell cycle and how cells respond to DNA damage. By using these tools, he and his colleagues have identified multiple genes involved in cell-cycle regulation.
Elledge’s work has earned him many awards, including a 2001 Paul Marks Prize for Cancer Research and a 2003 election to the National Academy of Sciences. In his Inaugural Article (1), published in this issue of PNAS, Elledge and his colleagues describe the function of Fbw7, a protein involved in controlling cell proliferation. These findings add to the growing cache of cell-cycle knowledge with implications for cancer research.
During his junior year abroad at the University of Southampton in England, Elledge gave biology a try by taking an introductory course and a semester of genetics. The classes sparked an interest, which he kept alive by taking a biochemistry class on his return to the United States. It was during his biochemistry lectures that Elledge first heard about recombinant DNA. “I just thought it was fabulous,” he said. “Once biology got down to being molecular, then it intersected with my interests.”
After receiving his bachelor’s degree in 1978, Elledge applied to graduate programs in biology and chemistry. Although he had not yet decided on which field to focus, he chose to continue his studies at the Massachusetts Institute of Technology (MIT) Biology Department. “I didn’t know what I wanted to do, but they had a lot of people, so I figured I’d be able to sort it out,” he said. Elledge ended up working with bacterial geneticist Graham Walker. For his thesis, Elledge studied the error-prone DNA repair mechanism in Escherichia coli called SOS mutagenesis. His work identified and described the regulation of a group of enzymes now know as errorprone polymerases, the first members of which were the umuCD genes in E. coli (2–4).
Elledge’s schedule at MIT allowed him time for side projects, and he used the opportunity to develop a new cloning tool. His creation was spurred by the frustration of unsuccessfully trying to use two existing tools, lambda phage and bacterial plasmid libraries, to clone the umuC gene, which produces proteins necessary for UV and chemical mutagenesis in E. coli. By combining the tools, Elledge invented a technique that allowed him to approach future cloning problems of this type with great rapidity (5). With the new technique, “you could make large libraries in lambda that behave like plasmids. We called them `phasmid’ vectors, like plasmid and phage together,” said Elledge. The phasmid cloning method was an early cornerstone for molecular biology research.
In 1984, Elledge began a postdoctoral fellowship at Stanford University (Stanford, CA) with mentor Ronald Davis. “Davis is an inventor,” said Elledge. “We had a lot in common because I’m interested in developing new technologies and so is he.”
Elledge soon began working on homologous recombination, an important niche in the field of eukaryotic genetics. Working with the yeast genome, Elledge searched for rec A, a gene that allows DNA to recombine homologously. Although he never located rec A, his work accidentally led him to a family of genes known as ribonucleotide reductases (RNRs), which are involved in DNA production (6). Rec A and RNRs share the same last 4 amino acids, which caused an antibody crossreaction in one of Elledge’s experiments. Initially disappointed with the false positives in his hunt for rec A, Elledge was later delighted with his luck. He found that RNRs are turned on by DNA damage (6), and that these genes are regulated by the cell cycle (7). “It was just serendipity,” he said.
Elledge’s work in this area led to a job offer from Baylor College of Medicine, Houston, in 1989. Prior to leaving Stanford, Elledge attended a talk at the University of California, San Francisco, by Paul Nurse, a leader in cell-cycle research who would later win the 2001 Nobel Prize in medicine. Nurse described his success in isolating the homolog of a key human cell-cycle kinase gene, Cdc2, by using a mutant strain of yeast (8). Although Nurse’s methods were primitive, Elledge was struck by the message he carried: that cell-cycle regulation was functionally conserved, and that many human genes could be isolated by looking for complimentary genes in yeast. Elledge then took advantage of his past successes in building phasmid vectors to build a versatile human cDNA library that could be expressed in yeast.
In his first experiments after setting up a laboratory at Baylor, he introduced this library into yeast, screening for complimentary cell-cycle genes. He quickly identified the same Cdc2 gene isolated by Nurse. However, Elledge also discovered a related gene known as Cdk2. Elledge subsequently found that Cdk2 controlled the G1 to S cell-cycle transition, a step that often goes awry in cancer. These results were published in theEMBO Journal in 1991 (9). “It was one of the biggest papers I’ve had,” said Elledge.
Elledge also continued to capitalize on his unexpected discovery of RNRs and used them to perform genetic screens to identify genes involved in sensing and responding to DNA damage. He subsequently worked out the signal transduction pathways in both yeast and humans that recognize damaged DNA and replication problems (10–12). These “checkpoint” pathways are central to the prevention of genomic instability and a key to understanding tumorigenesis.
Elledge’s research caught the attention of Wade Harper, a new member of Baylor’s biochemistry faculty. Combining their efforts, Harper and Elledge studied the regulation of Cdk2. “I was a geneticist and Wade was a biochemist. Together we were able to accomplish much more than either alone,” said Elledge. Elledge revamped a method for detecting protein interactions, known as the “two-hybrid system,” into a cloning method by combining it with his lambda cloning techniques. By using the new method, Harper and Elledge succeeded in isolating a gene known as p21, which they later identified as part of a family of Cdk2inhibitors. The gene also was cloned by Bert Vogelstein’s laboratory at Johns Hopkins University (Baltimore, MD), who discovered p21 was regulated by the cancer gene p53. Elledge and Vogelstein realized the similarity of their findings after chatting on the phone and published articles back-to-back in Cell in 1993 (13,14).
Elledge and his laboratory continued to look for other human genes that complimented yeast cell-cycle regulators. In 1996, his team identified a conserved motif, the F-box, that is present in some proteins. This motif recognizes specific protein sequences and tags them with ubiquitin for destruction. The buildup of certain proteins can sabotage the cell cycle and bring it to a halt; thus, destroying these proteins keeps cells dividing. Further investigation showed that the F-box sequence is ubiquitous throughout evolution. “There were so many F-box proteins that we figured it was going to be very central,” he said. Since Elledge’s laboratory published its first article on the F-box in 1998 (15), almost a thousand articles have reported investigations of F-box proteins and related ubiquitin ligases. The F-box has been implicated in numerous pathways, including gene expression, the immune response, cell morphology, cancer, and circadian rhythms.
Elledge’s focus still centers on the F-box motif and the roles played by its multitude of variations. In his Inaugural Article, found on page 3338, Elledge and his colleagues (1) describe mouse knockouts missing the gene to create an F-box protein known as Fbw7. Previous research suggested that Fbw7 controls the degradation of cyclin E, a protein that drives cell proliferation. By studying the knockouts, Elledge’s team showed that Fbw7 controls not only the abundance of cyclin E but also Notch protein. Both of these proteins play key roles in regulating mammalian development.
Elledge’s findings add to the growing body of knowledge on how F-box proteins operate in cells. However, with the function of hundreds of different F-box proteins currently unknown, Elledge and his collaborators, including Wade Harper, will have their work cut out for decades more. He and his laboratory plan to continue studying the genetics and genomics of different F-box proteins, elucidating their roles in cell proliferation. Elledge expects that this vast mystery, combined with his regular discoveries, will keep his passion alive. “I’m a scientist. I want to discover new things, and I want to develop new ways of looking at things. That’s what makes me excited, and that’s what I’m interested in,” he said.
This is a Biography of a recently elected member of the National Academy of Sciences to accompany the member’s Inaugural Article on page 3338.
References
Tetzlaff, M. T., Yu, W., Li, M., Zhang, P., Finegold, M., Mahon, K., Harper, J. W., Schwartz, R. J. & Elledge, S. J. (2004) Proc. Natl. Acad. Sci. USA 100 , 3338-3345.
Elledge, S. J. & Walker, G. C. (1983) J. Mol. Biol. 164 , 175-192.
- Elledge, S. J. & Walker, G. C. (1983) J. Bacteriol. 155 , 1306-1315.
Perry, K., Elledge, S. J., Mitchell, B. B., Marsh, L. & Walker, G. C. (1985) Proc. Natl. Acad. Sci. USA 82 , 4331-4335.
Elledge, S. J. & Walker, G. C. (1985) J. Bacteriol. 162 , 777-783.
Elledge, S. J. & Davis, R. W. (1987) Mol. Cell. Biol. 7 , 2783-2793.
Elledge, S. J. & Davis, R.W. (1990) Genes Dev. 4, 740-751.
Lee, M. G. & Nurse, P. (1987) Nature 32 , 31-35.
Elledge, S. & Spottswood, M. (1991) EMBO J. 10 , 2653-2659.
Zhou, Z. & Elledge, S. J. (1993) Cell 75, 1119-1127.
- Allen, J. B., Zhou, Z., Siede, W., Friedberg, E. C. & Elledge, S. J. (1994) Genes Dev. 8, 2401-2415.
Zou, L. & Elledge, S. J. (2003) Science 300, 1542-1548.
Harper, J. W., Adami, G., Wei, N., Keyomarsi, K. & Elledge, S. J. (1993) Cell 75 , 805-816.
El-Deiry, W. S., Tokino, T., Velculescu, V. E., Levy, D. B., Parsons, R., Trent, J. M., Lin, D., Mercer, W. E., Kinzler, K. W. & Vogelstein, B. (1993) Cell 75, 817-825.
Bai, C., Sen, P., Hofmann, K., Ma, L., Goebl, M., Harper, J.W. & Elledge, S. J. (1996) Cell 86, 263-274.
http://news.harvard.edu/gazette/story/2015/09/geneticist-stephen-j-elledge-receives-lasker-award/
The DNA Damage Response—Self-awareness for DNAThe 2015 Albert Lasker Basic Medical Research Award
Novel Protein May Open Door to New Therapies for Infection and Cancer
GEN News Aug 28, 2015 http://www.genengnews.com/gen-news-highlights/novel-protein-may-open-door-to-new-therapies-for-infection-and-cancer/81251675/
Scientists at Florida State University say they have taken a critical step forward in the fight against cancer with a discovery that could open up the door for new research and treatment options.
Fanxiu Zhu, Ph.D., the FSU Margaret and Mary Pfeiffer Endowed Professor for Cancer Research, and his team uncovered a viral protein in the cell that inhibits the major DNA sensor and thus the body’s response to viral infection, suggesting that this cellular pathway could be manipulated to help a person fight infection, cancer, or autoimmune diseases. They named the protein KicGas.
“We can manipulate the protein and/or the sensor to boost or tune down the immune response in order to fight infectious and autoimmune diseases, as well as cancers,” said Dr. Zhu, whose study (“Inhibition of cGAS-cGAMP DNA-Sensing Signaling by a Herpesvirus Virion Protein”) was published in Cell Host and Microbe.
Dr. Zhu leads a research team investigating how DNA viruses can cause cancer. About 15% of human cancer cases are caused by viruses, so scientists have been seeking answers about how the body responds to viral infection and how some viruses maintain life-long infections.
In the past few years, researchers finally identified the major DNA sensor in cells, known as cGas. That spurred researchers to further examine this sensor in the context of human disease because ideally that sensor should have been alerting the body to fight disease brought by a DNA virus.
Although people are equipped with sophisticated immune systems to cope with viral infection, many viruses have co-evolved mechanisms to evade or suppress the body’s immune responses. So the discovery of this protein is critical to further exploration of how these DNA viruses work and how they can be thwarted.
To uncover this protein, Dr. Zhu’s team studies Kaposi’s sarcoma-associated herpesvirus (KSHV), a human herpesvirus that causes some forms of lymphoma and Kaposi’s sarcoma, a cancer commonly occurring in AIDS patients and other immunocompromised individuals.
In this study, researchers screened every protein in a KSHV cell (90 in total) and ultimately found that one of them directly inhibited the DNA sensor called cGAS. They infected human cell lines with the Kaposi’s sarcoma virus to mimic natural infection, and found when they eliminated the inhibitor protein (KicGas) the cells produced a much stronger immune response.
To do this work, Dr. Zhu collaborated with several scientists both in the U.S. and Germany, including Hong Li, Ph.D., FSU professor of chemistry and biochemistry.
Dr. Li, whose focuses are molecular biology and molecular biophysics, specifically examined how the protein inhibited the cGAS activity in test tubes. For the next phase of research, she is building a three-dimensional model of the interactions to help them better understand how the inhibitor functions.
“These are hard problems to solve, and there is still much to learn here,” Dr. Li said. Learning how the inhibitor functions is a big next step, though. “Once we figure that out, we can hopefully design something to fight the disease,” according to Dr. Zhu.
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