PROFILE
PROFILE
Profile of Graham C. Walker Jennifer Viegas Science Writer
When biologist Graham Walker was introduced to DNA during a biology course at Carleton University in Canada, it was fascination at first sight. “I thought DNA was the coolest molecule in the world and decided I wanted to work on it,” says the recently elected National Academy of Sciences member. Since then, Walker’s research has helped explicate the structure and function of proteins involved in DNA repair and mutagenesis, with potential applications for cancer treatments and antibiotics. His work has also contributed to our understanding of how bacteria infect plants and mammals. For nearly four decades Walker has taught at the Massachusetts Institute of Technology (MIT), where he is in charge of the biology undergraduate program. “I am proud of having been able to make my research contributions at the same time that I was devoting considerable time and effort towards improving education and helping undergraduate students,” he says. Since 2002, Walker has been a Howard Hughes Medical Institute Professor and used his funding to establish a science education group with meetings modeled on those of his research laboratory. The group’s accomplishments include contributing to the internationally used biology education software programs StarBiochem and StarGenetics. Science vs. Classics
Walker’s interest in science blossomed when he was a child, spending summers in the Canadian woods at his family’s rustic cabin on the shore of the Gatineau River, north of Ottawa, Canada. “There were so many things I could see and understand about biology by just keeping my eyes open as I wandered through the forest and along the edge of the water,” he says. The chemistry set given to him by his biochemist mother as well as Isaac Asimov’s Building Blocks of the Universe inspired Walker to conduct scientific experiments at home. As a young student, Walker excelled in Latin and the classics, but decided to focus on science. In 1966, Walker went to Carleton University in his hometown of Ottawa, hoping to study quantum mechanics as a chemistry major. The introductory biology class on www.pnas.org/cgi/doi/10.1073/pnas.1400519111
DNA, however, changed those plans. “I knew just enough about the state of quantum mechanics in 1966 to realize that it wasn’t quite ready to deal with a molecule as big as DNA, so I decided to become an organic chemist instead so that I could synthesize it,” he says. Focus on Nucleic Acids
Carleton chemistry professor Robert Wightman was an early, influential mentor. As Walker says, “He not only ignited the fire in my belly for experimentation by letting me synthesize a methylated nucleoside in his lab for my honors thesis, but tolerated with good humor the large explosion that occurred when my reaction generating diazomethane got out of control.” Wightman arranged for Walker to work in the laboratory of molecular biologist Saran Narang at the National Research Council in Ottawa. Narang’s group at the time competed with another group, led by Walker’s future MIT colleague H. Gobind Khorana, to synthesize the first gene. Walker was excited by the prospects and decided to go to graduate school in chemistry to study nucleic acids. Wightman introduced Walker to the work of organic chemist Nelson Leonard at the University of Illinois. Leonard became Walker’s doctorate mentor, as did biochemist Olke Uhlenbeck. “I soon found myself running back and forth excitedly between their two labs, developing a half-chemical, half-enzymatic way of synthesizing short oligoribonucleotides [fragments of RNA] that had previously been unattainable,” Walker recalls. He later used the enzyme T4 RNA ligase, which he had purified, to carry out the intermolecular joining of two RNA oligonucleotides. “Think Like a Cell”
By the end of his graduate work, Walker realized that he did not wish to focus exclusively on nucleic acid chemistry or biochemistry. “Rather, I wanted to be able to pursue interesting biological problems using whatever experimental approach was required,” he says. “With that in mind, I decided I needed to gain experience with living cells and genetics.” Those considerations
Graham C. Walker. Image credit: Jan Walker.
led Walker to the laboratory of microbial geneticist and biochemist Bruce Ames at the University of California at Berkeley. Walker admired Ames’ understanding of bacterial physiology that allowed him to “think like a cell,” as Walker puts it. From 1974 to 1976, Walker’s postdoctoral work with Ames led to findings concerning chemical and radiation mutagenesis. For example, Walker used his budding skills as a bacterial geneticist to identify the mutagenesis-enhancing genes on pKM101, a derivative of a naturally occurring drug-resistant plasmid. Ames’ group created the “Ames test,” still used today, to test whether a given chemical is likely to cause cancer. The presence of the plasmid pKM101 in the bacterial tester strains increases error-prone repair of DNA damage. Walker says, “I learned ways to think about bacterial physiology from Bruce and was struck by his ability to connect superficially unrelated concepts from different fields to gain new insights.” This is a Profile of a recently elected member of the National Academy of Sciences to accompany the member’s Inaugural Article on page 3217.
PNAS | March 4, 2014 | vol. 111 | no. 9 | 3201–3202
DNA Damage and the SOS System
In 1976, Walker was offered an assistant professorship in the MIT Department of Biology that he eagerly accepted. He credits Boris Magasanik, Gene Brown, Salvador Luria, Maury Fox, David Botstein, Mary-Lou Pardue, Lisa Steiner, Tom RajBhandary, and Harvey Lodish as his early mentors at MIT. Walker’s prior research on the mutagenesis-enhancing function of pKM101 led to the analysis of Escherichia coli’s “SOS system,” a set of physiological responses induced by DNA damage. By screening E. coli derivatives carrying random fusions, Walker and his graduate student Cynthia Kenyon were able to identify DNA damage-inducible genes that are regulated as part of E. coli’s SOS response (1). Many of the genes encode functions involved in DNA repair or mutagenesis. “These results constituted the first direct evidence, in any organism, that DNA damage induces the expression of a set of genes,” Walker says. Walker later showed that the mutagenesisenhancing genes on pKM101 were orthologs of the chromosomal umuDC genes, required for most UV and chemical mutagenesis in E. coli and cloned by his graduate student Stephen Elledge (2). Additional studies shed further light on proteins associated with these genes and the SOS system (3, 4). More recent work identified the biological role of one of the most common DNA-damage response enzymes, DinB (5), and suggested an additional mechanism by which antibiotics can become toxic to bacterial cells (6). Formulating Cancer Treatment Strategies
After being named an American Cancer Society Research Professor in 2002, Walker collaborated with MIT colleague Michael Hemann. Together, they showed that diminishing the activity of a DNA repair pathway by reducing the activity of the specialized enzyme Rev1 dramatically reduced the frequency of acquired drug resistance when tumors relapse after chemotherapy (7). Interfering with the same pathway by reducing the level of the protein Rev3 made resistant lung cancers sensitive to the chemotherapy drug cisplatin (8). Walker and his colleagues suggested that interfering with the pathway might improve the effects of chemotherapy. Recently, he and his collaborators used those insights to make nanoparticles that prevented tumor growth in a human lymph node carcinoma of the prostate gland in a mouse model (9).
3202 | www.pnas.org/cgi/doi/10.1073/pnas.1400519111
One Bacterium, Many Research Applications
Another primary focus of Walker’s research concerns Rhizobium-legume symbiosis. During Walker’s postdoctoral years, recombinant DNA technology was developed and transposons, which are DNA sequences that can change position within the genome, routinely began to be used in bacterial genetics. “I realized these advances made it possible, in principle, to develop an E. coli-style genetic system for any bacterium and began to study Sinorhizobium meliloti, which forms a nitrogen-fixing symbiosis with its legume hosts,” Walker says. When teaching an undergraduate research laboratory, Walker unexpectedly discovered that the microbially produced polysaccharide succinoglycan is critical for legume nodule invasion (10). The way that S. meliloti infects cells turned out to have commonalities with how the cattle disease-causing Brucella abortus establishes chronic infection (11). Studies of symbiotically defective S. meliloti mutants additionally led to the discovery of the longsought “missing step” in vitamin B12 biosynthesis (12) and of a previously unrecognized ribonuclease enzyme, YbeY (13), which has a central role in RNA metabolism. Paving the Way for a New Class of Antibiotics
Walker’s Inaugural Article, “Global analysis of cell cycle gene expression of the legume symbiont Sinorhizobium meliloti”
1 Kenyon CJ, Walker GC (1980) DNA-damaging agents stimulate gene expression at specific loci in Escherichia coli. Proc Natl Acad Sci USA 77(5):2819–2823. 2 Elledge SJ, Walker GC (1983) Proteins required for ultraviolet light and chemical mutagenesis. Identification of the products of the umuC locus of Escherichia coli. J Mol Biol 164(2):175–192. 3 Perry KL, Elledge SJ, Mitchell BB, Marsh L, Walker GC (1985) umuDC and mucAB operons whose products are required for UV light- and chemical-induced mutagenesis: UmuD, MucA, and LexA proteins share homology. Proc Natl Acad Sci USA 82(13):4331–4335. 4 Nohmi T, Battista JR, Dodson LA, Walker GC (1988) RecAmediated cleavage activates UmuD for mutagenesis: Mechanistic relationship between transcriptional derepression and posttranslational activation. Proc Natl Acad Sci USA 85(6): 1816–1820. 5 Jarosz DF, Godoy VG, Delaney JC, Essigmann JM, Walker GC (2006) A single amino acid governs enhanced activity of DinB DNA polymerases on damaged templates. Nature 439(7073):225–228. 6 Foti JJ, Devadoss B, Winkler JA, Collins JJ, Walker GC (2012) Oxidation of the guanine nucleotide pool underlies cell death by bactericidal antibiotics. Science 336(6079):315–319. 7 Xie K, Doles J, Hemann MT, Walker GC (2010) Error-prone translesion synthesis mediates acquired chemoresistance. Proc Natl Acad Sci USA 107(48):20792–20797. 8 Doles J, et al. (2010) Suppression of Rev3, the catalytic subunit of Pol{zeta}, sensitizes drug-resistant lung tumors to chemotherapy. Proc Natl Acad Sci USA 107(48):20786–20791.
(14) is published with the accompanying article, “Host plant peptides elicit a transcriptional response to control the Sinorhizobium meliloti cell cycle during symbiosis” (15). Together, these reports shed light on plantbacterium symbiosis. Walker and his team carried out the first analysis of the S. meliloti cell cycle and showed that a particular plantencoded peptide, NCR247, affects cell cycle regulation, cell division, chromosome segregation genes, and certain signaling pathways important for symbiosis. The discovery sheds light on the molecular strategies underlying certain chronic intracellular pathogens of plants and animals. The findings, along with other research on symbiosis, could lead to a new class of broad-spectrum antibiotics to treat infectious diseases. For his contributions, Walker was awarded fellowship in the American Academy of Arts and Sciences in 2004. He has also received the Environmental Mutagen Society Award, along with many other honors. In future, Walker says, “I plan to continue studying how cells respond to DNA damage and the molecular mechanisms underlying the Rhizobium–legume symbiosis, but hope that the discovery-based component of my research style will continue to lead to exciting unanticipated insights into fundamental cellular processes. I also hope that some of our work may impact human health by improving chemotherapy and by helping us to address the impending antibiotic crisis.”
9 Xu X, et al. (2013) Enhancing tumor cell response to chemotherapy through nanoparticle-mediated codelivery of siRNA and cisplatin prodrug. Proc Natl Acad Sci USA 110(46): 18638–18643. 10 Leigh JA, Signer ER, Walker GC (1985) Exopolysaccharidedeficient mutants of Rhizobium meliloti that form ineffective nodules. Proc Natl Acad Sci USA 82(18):6231–6235. 11 LeVier K, Phillips RW, Grippe VK, Roop RM, 2nd, Walker GC (2000) Similar requirements of a plant symbiont and a mammalian pathogen for prolonged intracellular survival. Science 287(5462): 2492–2493. 12 Taga ME, Larsen NA, Howard-Jones AR, Walsh CT, Walker GC (2007) BluB cannibalizes flavin to form the lower ligand of vitamin B12.. Nature 446(7134):449–453. 13 Jacob AI, Köhrer C, Davies BW, RajBhandary UL, Walker GC (2013) Conserved bacterial RNase YbeY plays key roles in 70S ribosome quality control and 16S rRNA maturation. Mol Cell 49(3): 427–438. 14 Walker GC (2014) Global analysis of cell cycle gene expression of the legume symbiont Sinorhizobium meliloti. Proc Natl Acad Sci USA 111:3217–3224. 15 Penterman J, et al. (2014) Host plant peptides elicit a transcriptional response to control the Sinorhizobium meliloti cell cycle during symbiosis. Proc Natl Acad Sci USA 111:3561–3566.
Viegas
PROFILE
Profile of Graham C. Walker Jennifer Viegas Science Writer
When biologist Graham Walker was introduced to DNA during a biology course at Carleton University in Canada, it was fascination at first sight. “I thought DNA was the coolest molecule in the world and decided I wanted to work on it,” says the recently elected National Academy of Sciences member. Since then, Walker’s research has helped explicate the structure and function of proteins involved in DNA repair and mutagenesis, with potential applications for cancer treatments and antibiotics. His work has also contributed to our understanding of how bacteria infect plants and mammals. For nearly four decades Walker has taught at the Massachusetts Institute of Technology (MIT), where he is in charge of the biology undergraduate program. “I am proud of having been able to make my research contributions at the same time that I was devoting considerable time and effort towards improving education and helping undergraduate students,” he says. Since 2002, Walker has been a Howard Hughes Medical Institute Professor and used his funding to establish a science education group with meetings modeled on those of his research laboratory. The group’s accomplishments include contributing to the internationally used biology education software programs StarBiochem and StarGenetics. Science vs. Classics
Walker’s interest in science blossomed when he was a child, spending summers in the Canadian woods at his family’s rustic cabin on the shore of the Gatineau River, north of Ottawa, Canada. “There were so many things I could see and understand about biology by just keeping my eyes open as I wandered through the forest and along the edge of the water,” he says. The chemistry set given to him by his biochemist mother as well as Isaac Asimov’s Building Blocks of the Universe inspired Walker to conduct scientific experiments at home. As a young student, Walker excelled in Latin and the classics, but decided to focus on science. In 1966, Walker went to Carleton University in his hometown of Ottawa, hoping to study quantum mechanics as a chemistry major. The introductory biology class on www.pnas.org/cgi/doi/10.1073/pnas.1400519111
DNA, however, changed those plans. “I knew just enough about the state of quantum mechanics in 1966 to realize that it wasn’t quite ready to deal with a molecule as big as DNA, so I decided to become an organic chemist instead so that I could synthesize it,” he says. Focus on Nucleic Acids
Carleton chemistry professor Robert Wightman was an early, influential mentor. As Walker says, “He not only ignited the fire in my belly for experimentation by letting me synthesize a methylated nucleoside in his lab for my honors thesis, but tolerated with good humor the large explosion that occurred when my reaction generating diazomethane got out of control.” Wightman arranged for Walker to work in the laboratory of molecular biologist Saran Narang at the National Research Council in Ottawa. Narang’s group at the time competed with another group, led by Walker’s future MIT colleague H. Gobind Khorana, to synthesize the first gene. Walker was excited by the prospects and decided to go to graduate school in chemistry to study nucleic acids. Wightman introduced Walker to the work of organic chemist Nelson Leonard at the University of Illinois. Leonard became Walker’s doctorate mentor, as did biochemist Olke Uhlenbeck. “I soon found myself running back and forth excitedly between their two labs, developing a half-chemical, half-enzymatic way of synthesizing short oligoribonucleotides [fragments of RNA] that had previously been unattainable,” Walker recalls. He later used the enzyme T4 RNA ligase, which he had purified, to carry out the intermolecular joining of two RNA oligonucleotides. “Think Like a Cell”
By the end of his graduate work, Walker realized that he did not wish to focus exclusively on nucleic acid chemistry or biochemistry. “Rather, I wanted to be able to pursue interesting biological problems using whatever experimental approach was required,” he says. “With that in mind, I decided I needed to gain experience with living cells and genetics.” Those considerations
Graham C. Walker. Image credit: Jan Walker.
led Walker to the laboratory of microbial geneticist and biochemist Bruce Ames at the University of California at Berkeley. Walker admired Ames’ understanding of bacterial physiology that allowed him to “think like a cell,” as Walker puts it. From 1974 to 1976, Walker’s postdoctoral work with Ames led to findings concerning chemical and radiation mutagenesis. For example, Walker used his budding skills as a bacterial geneticist to identify the mutagenesis-enhancing genes on pKM101, a derivative of a naturally occurring drug-resistant plasmid. Ames’ group created the “Ames test,” still used today, to test whether a given chemical is likely to cause cancer. The presence of the plasmid pKM101 in the bacterial tester strains increases error-prone repair of DNA damage. Walker says, “I learned ways to think about bacterial physiology from Bruce and was struck by his ability to connect superficially unrelated concepts from different fields to gain new insights.” This is a Profile of a recently elected member of the National Academy of Sciences to accompany the member’s Inaugural Article on page 3217.
PNAS | March 4, 2014 | vol. 111 | no. 9 | 3201–3202
DNA Damage and the SOS System
In 1976, Walker was offered an assistant professorship in the MIT Department of Biology that he eagerly accepted. He credits Boris Magasanik, Gene Brown, Salvador Luria, Maury Fox, David Botstein, Mary-Lou Pardue, Lisa Steiner, Tom RajBhandary, and Harvey Lodish as his early mentors at MIT. Walker’s prior research on the mutagenesis-enhancing function of pKM101 led to the analysis of Escherichia coli’s “SOS system,” a set of physiological responses induced by DNA damage. By screening E. coli derivatives carrying random fusions, Walker and his graduate student Cynthia Kenyon were able to identify DNA damage-inducible genes that are regulated as part of E. coli’s SOS response (1). Many of the genes encode functions involved in DNA repair or mutagenesis. “These results constituted the first direct evidence, in any organism, that DNA damage induces the expression of a set of genes,” Walker says. Walker later showed that the mutagenesisenhancing genes on pKM101 were orthologs of the chromosomal umuDC genes, required for most UV and chemical mutagenesis in E. coli and cloned by his graduate student Stephen Elledge (2). Additional studies shed further light on proteins associated with these genes and the SOS system (3, 4). More recent work identified the biological role of one of the most common DNA-damage response enzymes, DinB (5), and suggested an additional mechanism by which antibiotics can become toxic to bacterial cells (6). Formulating Cancer Treatment Strategies
After being named an American Cancer Society Research Professor in 2002, Walker collaborated with MIT colleague Michael Hemann. Together, they showed that diminishing the activity of a DNA repair pathway by reducing the activity of the specialized enzyme Rev1 dramatically reduced the frequency of acquired drug resistance when tumors relapse after chemotherapy (7). Interfering with the same pathway by reducing the level of the protein Rev3 made resistant lung cancers sensitive to the chemotherapy drug cisplatin (8). Walker and his colleagues suggested that interfering with the pathway might improve the effects of chemotherapy. Recently, he and his collaborators used those insights to make nanoparticles that prevented tumor growth in a human lymph node carcinoma of the prostate gland in a mouse model (9).
3202 | www.pnas.org/cgi/doi/10.1073/pnas.1400519111
One Bacterium, Many Research Applications
Another primary focus of Walker’s research concerns Rhizobium-legume symbiosis. During Walker’s postdoctoral years, recombinant DNA technology was developed and transposons, which are DNA sequences that can change position within the genome, routinely began to be used in bacterial genetics. “I realized these advances made it possible, in principle, to develop an E. coli-style genetic system for any bacterium and began to study Sinorhizobium meliloti, which forms a nitrogen-fixing symbiosis with its legume hosts,” Walker says. When teaching an undergraduate research laboratory, Walker unexpectedly discovered that the microbially produced polysaccharide succinoglycan is critical for legume nodule invasion (10). The way that S. meliloti infects cells turned out to have commonalities with how the cattle disease-causing Brucella abortus establishes chronic infection (11). Studies of symbiotically defective S. meliloti mutants additionally led to the discovery of the longsought “missing step” in vitamin B12 biosynthesis (12) and of a previously unrecognized ribonuclease enzyme, YbeY (13), which has a central role in RNA metabolism. Paving the Way for a New Class of Antibiotics
Walker’s Inaugural Article, “Global analysis of cell cycle gene expression of the legume symbiont Sinorhizobium meliloti”
1 Kenyon CJ, Walker GC (1980) DNA-damaging agents stimulate gene expression at specific loci in Escherichia coli. Proc Natl Acad Sci USA 77(5):2819–2823. 2 Elledge SJ, Walker GC (1983) Proteins required for ultraviolet light and chemical mutagenesis. Identification of the products of the umuC locus of Escherichia coli. J Mol Biol 164(2):175–192. 3 Perry KL, Elledge SJ, Mitchell BB, Marsh L, Walker GC (1985) umuDC and mucAB operons whose products are required for UV light- and chemical-induced mutagenesis: UmuD, MucA, and LexA proteins share homology. Proc Natl Acad Sci USA 82(13):4331–4335. 4 Nohmi T, Battista JR, Dodson LA, Walker GC (1988) RecAmediated cleavage activates UmuD for mutagenesis: Mechanistic relationship between transcriptional derepression and posttranslational activation. Proc Natl Acad Sci USA 85(6): 1816–1820. 5 Jarosz DF, Godoy VG, Delaney JC, Essigmann JM, Walker GC (2006) A single amino acid governs enhanced activity of DinB DNA polymerases on damaged templates. Nature 439(7073):225–228. 6 Foti JJ, Devadoss B, Winkler JA, Collins JJ, Walker GC (2012) Oxidation of the guanine nucleotide pool underlies cell death by bactericidal antibiotics. Science 336(6079):315–319. 7 Xie K, Doles J, Hemann MT, Walker GC (2010) Error-prone translesion synthesis mediates acquired chemoresistance. Proc Natl Acad Sci USA 107(48):20792–20797. 8 Doles J, et al. (2010) Suppression of Rev3, the catalytic subunit of Pol{zeta}, sensitizes drug-resistant lung tumors to chemotherapy. Proc Natl Acad Sci USA 107(48):20786–20791.
(14) is published with the accompanying article, “Host plant peptides elicit a transcriptional response to control the Sinorhizobium meliloti cell cycle during symbiosis” (15). Together, these reports shed light on plantbacterium symbiosis. Walker and his team carried out the first analysis of the S. meliloti cell cycle and showed that a particular plantencoded peptide, NCR247, affects cell cycle regulation, cell division, chromosome segregation genes, and certain signaling pathways important for symbiosis. The discovery sheds light on the molecular strategies underlying certain chronic intracellular pathogens of plants and animals. The findings, along with other research on symbiosis, could lead to a new class of broad-spectrum antibiotics to treat infectious diseases. For his contributions, Walker was awarded fellowship in the American Academy of Arts and Sciences in 2004. He has also received the Environmental Mutagen Society Award, along with many other honors. In future, Walker says, “I plan to continue studying how cells respond to DNA damage and the molecular mechanisms underlying the Rhizobium–legume symbiosis, but hope that the discovery-based component of my research style will continue to lead to exciting unanticipated insights into fundamental cellular processes. I also hope that some of our work may impact human health by improving chemotherapy and by helping us to address the impending antibiotic crisis.”
9 Xu X, et al. (2013) Enhancing tumor cell response to chemotherapy through nanoparticle-mediated codelivery of siRNA and cisplatin prodrug. Proc Natl Acad Sci USA 110(46): 18638–18643. 10 Leigh JA, Signer ER, Walker GC (1985) Exopolysaccharidedeficient mutants of Rhizobium meliloti that form ineffective nodules. Proc Natl Acad Sci USA 82(18):6231–6235. 11 LeVier K, Phillips RW, Grippe VK, Roop RM, 2nd, Walker GC (2000) Similar requirements of a plant symbiont and a mammalian pathogen for prolonged intracellular survival. Science 287(5462): 2492–2493. 12 Taga ME, Larsen NA, Howard-Jones AR, Walsh CT, Walker GC (2007) BluB cannibalizes flavin to form the lower ligand of vitamin B12.. Nature 446(7134):449–453. 13 Jacob AI, Köhrer C, Davies BW, RajBhandary UL, Walker GC (2013) Conserved bacterial RNase YbeY plays key roles in 70S ribosome quality control and 16S rRNA maturation. Mol Cell 49(3): 427–438. 14 Walker GC (2014) Global analysis of cell cycle gene expression of the legume symbiont Sinorhizobium meliloti. Proc Natl Acad Sci USA 111:3217–3224. 15 Penterman J, et al. (2014) Host plant peptides elicit a transcriptional response to control the Sinorhizobium meliloti cell cycle during symbiosis. Proc Natl Acad Sci USA 111:3561–3566.
Viegas
