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Academic Life: The Whole Package

2008; Elsevier BV; Volume: 283; Issue: 30 Linguagem: Inglês

10.1074/jbc.x800004200

1083-351X

Christopher K. Mathews,

ATP Synthase and ATPases Research

When I received the unexpected but welcome invitation to write a Reflections article, I wondered which aspects of my life in science are sufficiently distinctive to make the article worth reading. One aspect may be my survival as a department chair for nearly 25 years while struggling to contribute as well in teaching and research, in other words, embracing the “whole package” of academic life: teaching, research, and service. Another part may be my experience as coauthor of a biochemistry textbook. To deal with both, I will begin with my introduction to biochemistry. My nascent interest in biochemistry became full-blown during my junior year at Reed College, when I read Currents in Biochemical Research 1956, a compendium edited by David E. Green. The following summer, my grandparents took me on a tour of several East Coast graduate departments. It was a thrill to meet William McElroy at Johns Hopkins, John Buchanan at MIT, and Konrad Bloch at Harvard. However, when I visited Brookhaven National Laboratory, my host, Dr. Robert Steele, asked, “Why are you considering universities in the East when one of the best biochemistry departments in the country is right in your own back yard, in Seattle?” So I arranged a visit to the University of Washington, where I met, among others, Ed Krebs, who reminded me gently that he was not Krebs of the cycle. Who might have predicted that some 35 years later Ed Krebs would become as famous as Sir Hans when he and Eddie Fischer would share the Nobel Prize for discovering protein phosphorylation, work that was under way in Seattle at that very moment? During my first week in graduate school at Washington, someone asked about my research interests. “Something to do with enzymes that is biologically significant,” I replied, aligning myself with perhaps 90% of the 1958 cohort of entering biochemistry graduate students. The research of Dr. Frank Huennekens, on folate coenzymes and one-carbon metabolism, met my criteria. His laboratory had previously shown that folate antagonists such as Methotrexate act by inhibiting dihydrofolate reductase (DHFR). The idea of targeting drugs to treat disease was exciting. Frank suggested as an introductory project that I develop an enzymatic method for preparative-scale synthesis of the biologically active stereoisomer of tetrahydrofolate. This was easily done by treating dihydrofolate with DHFR and NADPH, followed by DEAE-cellulose column purification. When I had completed the project, I was surprised when Frank suggested that we write it up and send it to the Journal of Biological Chemistry. Several months later in November 1960, my first paper was published (1Mathews C.K. Huennekens F.M. J. Biol. Chem. 1960; 235: 3304-3308Abstract Full Text PDF PubMed Google Scholar), and again to my surprise, the paper was widely cited. In 1960, Erwin Chargaff came to Seattle for a series of lectures, which included some grumbling about people working with “columns of treated and mistreated cellulose.” Chargaff spoke at length about work done by “my student Seymour Cohen,” on the induction of new enzymes in bacteria infected with T-even bacteriophage (I later learned that Seymour had done his Ph.D. thesis with Chargaff on phospholipids and the phage work came much later). I was becoming interested in genetic biochemistry, and phage presented perhaps the best biological systems at that time. Reading Cohen's papers, I learned that the phage-induced enzymes discovered in his lab (thymidylate synthase and deoxycytidylate hydroxymethylase) used folate coenzymes. His laboratory provided an excellent link between my training with Frank and the more biological work I hoped to do. In January 1962, my wife, Kate (a former graduate school classmate), our infant son, Lawrence, and I enjoyed a snowy drive from Seattle to Philadelphia, where I began work in Seymour's lab at the University of Pennsylvania. My main project was a biochemical experiment to establish whether a phage-induced enzyme, dCMP hydroxymethylase, was synthesized de novo after T6 phage infection, as it must be if the induction of new enzyme activities in phage infection results from expression of phage genes. The experiment was to grow Escherichia coli in radiolabeled medium, centrifuge and wash the cells, transfer to cold medium, infect with phage, isolate the enzyme, and then demonstrate that it was non-radioactive, indicative of post-infection biosynthesis. An ambitious project, and it would never have been completed except that Seymour's lab was joined by Fred Brown, a distinguished British virologist who wished to learn some biochemical techniques. For the several months we worked together, Fred and I were totally immersed in the project, constantly exchanging ideas, criticisms, and banter. It was an exciting time for me, the more so because the project worked (2Mathews C.K. Brown F. Cohen S.S. J. Biol. Chem. 1964; 239: 2957-2963Abstract Full Text PDF PubMed Google Scholar). It mattered little to us that before our work was published John Buchanan's laboratory had described a class of temperature-sensitive T4 phage mutants that induced a heat-labile form of dCMP hydroxymethylase, establishing independently that the enzyme was a viral gene product. The intensity of the Mathews/Brown experience was heightened by the 4 days that we practically lived together, leaning upon each other for support, following the assassination of JFK. Joined by our second child, Anne, the four Mathews moved just before Christmas 1963 to New Haven, where I took up my first faculty position as Assistant Professor of Biology at Yale. It was a homecoming of sorts because my family had lived there from 1946 to 1949 while my father was on the faculty at Yale Medical School and before we all moved to the West Coast. My closest friend in the large and diverse Department of Biology was Gerry Wyatt, who had earlier worked with Seymour Cohen, when they discovered 5-hydroxymethylcytosine as a component of T-even phage DNAs. It was this finding that led to the discovery, a few years later, of phage-coded enzymes in Seymour's laboratory. Gerry had turned to insect biochemistry, but we shared numerous interests, and he was a great mentor. He attended my first several lectures, and afterward, he dissected each unmercifully. He also gave me some useful career hints, such as the importance, particularly in a diverse department where no one understands your research, of asking good questions in seminars. I had not realized that in some quarters seminar is a blood sport. For my independent research program, I wished to explore metabolic relationships between DNA precursors and DNA synthesis. Inasmuch as deoxyribonucleotides have no known biological functions except as DNA precursors, it seemed reasonable to expect closer coordination between the synthesis of deoxyribonucleoside triphosphates (dNTPs) and DNA replication than relations between ribonucleotides and transcription or amino acids and translation. T-even phage and their induced enzymes continued to be good systems for exploring ideas that I had developed in the Cohen laboratory. I was joined by two good graduate students and several good undergraduates, and things went reasonably well in the laboratory. I enjoyed my four years at Yale, but like all of my fellow assistant professors, I wondered about a future in which, according to the Yale Faculty Handbook, I would eventually be considered for promotion in competition with everyone in the world in my field and at my career stage. In my fourth year, when I was carrying Wyatt's teaching as well as my own during his sabbatical, my department chair told me that if I aspired to be a full professor at Yale, I would need to be recognized as one of the top fifty biologists in the country. With my heavy teaching and premedical advising duties, this seemed unrealistic. It was a relief, shortly thereafter, to receive a call from Don Hanahan, one of my professors in graduate school. Don had accepted the chairmanship of the Biochemistry Department at the new College of Medicine at the University of Arizona, and he was inviting me to join his department. Although I regretted the inconvenience to my two graduate students, who had to move with me to complete their Ph.D. theses, I jumped at the chance for total involvement in a new academic enterprise. We moved to Tucson in 1967, just in time to meet the first class of 32 medical students. I had enjoyed both my teaching and research at Yale, but I realized a much greater opportunity at Arizona to have an impact upon the institution. That is when I learned that I was in love with the whole package; whereas most professors can prosper by exhibiting excellence in one or two of the three realms of academic endeavor, I desired full engagement with all three. To succeed, of course, it was essential to sustain research productivity. With the help of some data brought from Yale plus experiments in my new lab, I published, in the spring of 1968, what I believe was the first research article from the University of Arizona College of Medicine (3Mathews C.K. J. Biol. Chem. 1968; 243: 5610-5615Abstract Full Text PDF PubMed Google Scholar). In early work at Arizona, we characterized the T4 phage-coded DHFR, which I had discovered as a postdoc. Graduate student John Erickson was the first to purify to homogeneity DHFR from any source by using an N-formylaminopterin affinity column. His spectrophotometric studies of Methotrexate binding to T4 DHFR suggested a model that was confirmed later through crystallographic studies in other laboratories. In other work, I was intrigued by the large number of phage gene products shown to be essential for DNA replication, but for which no biochemical function had been identified. In 1971, we knew that T4 gene 43 encoded DNA polymerase, whereas gene 32 encoded single-strand DNA-binding protein (SSB). Could any of the remaining dozen or so replication-related genes encode essential steps in dNTP biosynthesis? I measured dNTP pool sizes in cells infected by different T4 mutants. As expected, gene 42 mutants, defective in dCMP hydroxymethylase induction, could not accumulate 5-hydroxymethyl-dCTP, but all of the other mutants accumulated huge dNTP pools (with the mysterious exception of dGTP) (4Mathews C.K. J. Biol. Chem. 1972; 247: 7430-7438Abstract Full Text PDF PubMed Google Scholar). At the same time, Bruce Alberts and Nancy Nossal were taking the more direct and more productive approach, purifying the DNA-related gene products and identifying their biochemical activities (5Nossal N.G. Alberts B.M. Mathews C.K. Kutter E.M. Mosig G. Berget P.B. Bacteriophage T4. American Society for Microbiology, Washington, D. C.1983: 71-81Google Scholar). My initial appointment letter at Arizona identified me as an Associate Professor of Biochemistry (Temporary). In my second year, I noted that the “Temporary” had disappeared, with no explanation. I asked my chair, Don Hanahan, “Does this mean I have tenure?” He replied, “I'm glad to see that that went through.” A distinct contrast with today's 150-page promotion/tenure dossiers! It led me to fear that the University of Arizona was too lax in its standards, and I made myself a bit of a pest with my dean, expressing my concern that the College of Medicine would become fully tenured with mediocrities (present company excluded, of course). I worked also, along with Hanahan, to strengthen ties between our small medical school department and the biochemists on the main campus at Arizona. I sought opportunities to teach in main-campus courses. For universities to establish duplicate and competing departments on the same campus made little sense. Eventually, after both Hanahan and I had departed, Arizona did merge all biochemists into one strong University Department of Biochemistry, and I would like to think that Don and I, plus our other colleagues, sowed the seeds for this successful endeavor. Meanwhile in the laboratory, we began to wonder how the T4-infected cell is organized so that dNTP concentrations can be sustained at DNA replication sites in the face of enormous pool turnover, resulting from DNA chain growth rates of several hundred nucleotides per second. We confirmed that DNA polymerase is saturated with dNTPs in vivo. Data from both our laboratory and Bob Greenberg's suggested that dNTPs were somehow compartmentalized within T4-infected E. coli. How could this occur in a prokaryotic cell with no intracellular organelles? Graduate student Jim Summerton proposed a model of “functional compartmentation,” in which dNTPs near a replication fork have a much higher probability of incorporation into DNA than more remote dNTPs. Hence, the cell could contain two kinetically distinct dNTP pools: a replication-active pool, generated at replication sites and rapidly turned over, and an inactive pool, generated elsewhere, slowly replenished, but available for DNA repair. Similar ideas occurred to Greenberg (6Flanegan J.B. Greenberg G.R. J. Biol. Chem. 1977; 252: 3019-3027Abstract Full Text PDF PubMed Google Scholar), and for several years, our laboratories engaged in friendly competition, fortunately using different lines of evidence, which converged upon support for one model. In that model, a multienzyme complex, which we called “dNTP synthetase,” is juxtaposed with the replisome and maintains kinetically supported dNTP concentration gradients by shuttling intermediates from enzyme to enzyme within the complex, thereby maintaining saturating dNTP levels at the replisome. Graduate student Prem Reddy generated support for this model by finding that many of the T4 phage-coded enzymes cosedimented through sucrose gradients. About this time, I met Arthur Kornberg for the first time. When I told him of our work, he offered pithy advice: “You must be awfully careful how you interpret results from sucrose gradients.” This led us to characterize the enzyme aggregate kinetically; the aggregated enzymes catalyzed multistep reaction pathways far more efficiently than predicted for a model in which the enzymes do not interact. More important, when an enzyme not involved in the pathway under study was inactivated by means of a temperature-sensitive mutation, kinetic coupling among the pathway enzymes was abolished. Seymour Cohen communicated our first paper in this arena to the Proceedings of the National Academy of Sciences of the United States of America (7Reddy G.P.V. Singh A. Stafford M.E. Mathews C.K. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 3152-3156Crossref PubMed Scopus (37) Google Scholar), and several more followed. But those several more came from a new address. In 1977, I was invited to interview for the chairmanship of the Department of Biochemistry and Biophysics at Oregon State University. The prospect of moving to Corvallis, between Kate's home town (Berkeley, CA) and mine (Olympia, WA), was tempting. Even more tempting was the attraction of the 14-member faculty, all of whom were active in research, who shared adequate contiguous space, and who cherished an atmosphere of collegiality and mutual respect. The faculty told me that the chairmanship was a three-year re-electable term appointment, whereas the dean said, “Nonsense; you will serve at my pleasure.” Because a successful department chair must satisfy both bosses, the ambiguity did not concern me. Again, two important people were inconvenienced by our move: our son, who was finishing high school, and our daughter, who was beginning. We made arrangements ensuring that each would have all four years at the same high school and moved to Corvallis in January 1978. Those who remember Howard Schachman's famous Academic Metabolic Chart will recall that “department chair” is a relatively unstable intermediate. In a coupled reaction in which Friends are converted to Enemies, the Chair may be converted to Dean. More often, in a highly exergonic reaction, the Chair reverts to a professor without administrative responsibilities. Somehow, I fell into a deep free energy trough. Regularly, at three-year intervals, I submitted myself to the faculty for review, and regularly, I was returned for three more years as chair. Occasionally, the dean weighed in, but generally, the six deans and acting deans I worked with stayed out of the way. Two year-long sabbaticals in Sweden helped me retain my equilibrium, 1984–1985 with Peter Reichard and 1994–1995 with Britt-Marie Sjöberg. It is common for department chairs to grouse about the responsibilities and frustrations, and I did my share. But truth to tell, no one was holding a gun to my head to keep me in the job, so I must have found the satisfactions to outweigh the frustrations. At some point, one of my deans told me that he had always aspired to rise to the level of dean of a college of science. “Why didn't you aspire to a presidency if you wished to be an administrator?” I asked. “Because dean is the highest level at which you can spend most of your time with bright people,” he answered. “Funny,” I said, “I always thought that department chair was the level meeting that criterion.” My past chair, Don Hanahan, had taught me the importance of seeking outside funding to support the whole enterprise, not just the chair's own research. So I applied for an NIH training grant, and the first proposal netted a priority score of 318. On the pink sheet was written, “The training record of the faculty is not impressive; most of the graduates have taken positions in industry.” I protested to NIH and pointed out that Congress, which funds NIH, might not like to hear that NIH reviewers and administrators considered industrial careers to be second-rate. Our next several training grant applications were funded. Next, our administration asked me to write a large proposal to a regional foundation to support the upgrading of molecular genetics on the campus. “Yes, Chris,” said our president, “we have decided it is time to move in this exciting area.” Since the year was 1981, I said, “Yes, or maybe somewhat past time.” In any event, the application was funded, and we became one of the earlier universities to establish a multidepartmental center that supported molecular genetics through operation of a service laboratory, multidepartmental faculty searches, and memberships that provided faculty from mission-oriented departments (Horticulture, Food Science, Forestry, etc.) with a second intellectual home. As a potential chair, I saw in the Oregon State University Biochemistry/Biophysics Department several attractive features. First, the department had a real raison d'etre, with strong commitments to its graduate program, its undergraduate major program, and service responsibilities to nearly two dozen departments and programs elsewhere on our diverse Land Grant campus. Second, the department had strong links to those other departments and programs. About fifteen faculty members in sister departments held graduate faculty appointments, meaning that they could direct graduate student research in biochemistry/biophysics. Departmental faculty were frequently called upon for advice, sharing research equipment, and service on graduate committees. Third, our university is on the quarter system, which makes it easier to give a faculty member sole responsibility for a course and which also makes it easier for graduate students to select courses outside their own interest areas; a commitment for ten weeks is much less than for fifteen. It also meant that we could teach our introductory majors' biochemistry course by assigning a protein/enzyme chemist in Fall, a metabolic biochemist in Winter, and a genetic biochemist in Spring, giving majors and serious students three organized courses, each taught by an expert. All of these desirable features I did my best to keep in place. My continuing love affair with the whole package meant that I needed to contribute in my own teaching, and throughout my nearly 25 years as chair, I carried a substantial teaching load. I enjoyed sharing a graduate course in DNA-protein interactions with Ken van Holde. We evaluated students by having them write and defend original research proposals, a feature that was uncommon then, but widespread now. Also, I noticed that most faculty preferred not to teach in our year-long laboratory course. I think that thoughtful design of inquiry-based laboratory instruction is important, so I assigned myself to the lab course for several years. I am not sure that it changed my colleagues' attitudes, but at least they saw what the chair considered to be worthwhile. Another course that I continue to enjoy is “The News of Science,” a colloquium that I originated in our Honors College. The eight to twelve students in this course read Science magazine and select articles for oral presentation. All students are required to read each article to ensure a vigorous discussion after each talk, and students have been adventurous in selecting presentation topics. Our research activities moved in several directions, linked by common interests in relationships between dNTP metabolism and DNA replication. Graduate student Rick Bestwick established that mitochondria in mammalian cells contain dNTP pools that are physically and functionally distinct from the pools supplying nuclear DNA replication. Graduate student Sarla Purohit sequenced the T4 gene for DHFR at a time when sequencing 1 kb of DNA was much more than an afternoon's work, and she developed an excellent expression system for the gene. Graduate student Janet Leeds measured dNTP pools in mammalian cell nuclei and showed that the cells she studied do not exhibit a dNTP concentration gradient between the cytosol and nucleus. Postdoc Mary Slabaugh looked to vaccinia virus as a quasi-eukaryotic counterpart to the phage and bacterial systems we had worked with, and she discovered that vaccinia encodes a ribonucleotide reductase (RNR) that is closely related to all eukaryotic forms of this essential enzyme. Graduate student Steve Hendricks developed an assay for RNR that permits analysis of its four reactions in a single incubation mixture, and Steve and graduate student Korakod Chimploy used this assay to show that RNR is controlled both by concentrations of the four ribonucleotide substrates, competing for the same catalytic site, and by the allosteric mechanisms described elsewhere in kinetic studies with single substrates. Through it all, we continued to study the T4 dNTP synthetase enzyme complex we had discovered at Arizona and attempted to define its relationship to the T4 replisome. We were aided by work in our own lab and elsewhere, which allowed us to obtain all of the enzymes and proteins of interest to us as purified recombinant proteins. This permitted several approaches to analysis of the protein-protein interactions that stabilize the complex. For example, technician Linda Wheeler immobilized dCMP hydroxymethylase on a column and used two-dimensional gel electrophoresis to identify specifically retained T4 phage proteins. Application of this method to other enzymes in the complex revealed that the T4 gene 32 SSB bore an unexpectedly close relationship to enzymes of dNTP synthesis, and we proposed a role for the gp32 SSB in recruiting enzymes of the dNTP synthetase complex to the replisome (8Wheeler L.J. Ray N.B. Ungermann C. Hendricks S.P. Bernard M.A. Hanson E.S. Mathews C.K. J. Biol. Chem. 1996; 271: 11156-11162Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). At about the same time, graduate student Ralph Davis discovered a strong interaction between vaccinia virus RNR and a virus-coded SSB protein, suggesting a similar relationship for this large DNA virus. In 1987, Paul Srere organized the first Gordon Conference devoted to organization of metabolic sequences, and I found that facilitation of pathways by enzyme interactions was being seen in all areas of metabolism. Paul's own work on interactions among citric acid cycle enzymes inspired me to write this ditty. My goodness, Paul, you do babble on,And lead biochemical rabble onAbout your grand theoryThe Super Paul SrereTCA cycle metabolon Our interest in how dNTP enzymes are organized to sustain DNA synthesis rates led us to investigate also relationships between dNTP metabolism and DNA replication fidelity (9Mathews C.K. FASEB J. 2006; 20: 1300-1314Crossref PubMed Scopus (228) Google Scholar). Graduate student Geoff Sargent studied T4 phage mutants defective in their ability to induce phage-coded dCMP deaminase. He showed that cells infected by these mutant phage display greatly elevated pools of 5-hydroxymethyl-dCTP and diminished dTTP pools, as expected from the reaction catalyzed by dCMP deaminase. These mutants showed elevated spontaneous mutagenesis, with most of the mutations involving AT-to-GC transitions, as expected from the pool imbalance. Graduate students Xiaolin Zhang and Stella Martomo used an in vitro fidelity assay to investigate whether natural asymmetry in dNTP pools, with dGTP being under-represented, contributes toward error frequency in DNA replication. Stella found that balanced accumulation of dNTPs increases error rates in vitro, and Linda Wheeler and postdoc Indira Rajagopal found that the same occurs in vivo by overexpressing RNR to increase dNTPs proportionally in E. coli. This seems to result from a next-nucleotide effect, in which dNTP accumulations accelerate rates of chain elongation from DNA mismatches, essentially competing with exonucleolytic proofreading of the error. Meanwhile, our interest in mitochondrial dNTP metabolism was re-ignited by graduate student Shiwei Song. Shiwei made several notable observations (10Mathews C.K. Song S. FASEB J. 2007; 21: 2294-2303Crossref PubMed Scopus (30) Google Scholar), particularly his finding that dNTP pools in mitochondria from rat tissues are highly asymmetric, but in a different sense from whole-cell pools. Whereas pool measurements in whole cells or in nuclei show dGTP to represent only 5–10% of total dNTP, that proportion is as high as 90% in mitochondria from rat tissues. We do not yet know the significance or metabolic basis for this strong asymmetry, but in vitro fidelity assays suggest that it contributes toward the high mutation rate known for the mitochondrial genome. Moreover, the high mitochondrial dGTP concentration (as high as 100 μm), coupled with the oxidizing environment in the organelle, suggested that oxidation of this nucleotide might contribute to mutagenesis via incorporation of 8-oxo-dGTP into DNA opposite template adenine, thereby initiating a transversion mutation. Recent direct analyses of mitochondrial 8-oxo-dGTP pools suggest this indeed to be the case (11Pursell Z.F. McDonald J.T. Mathews C.K. Kunkel T.A. Nucleic Acids Res. 2008; 36: 2174-2181Crossref PubMed Scopus (65) Google Scholar). Our department teaches three different introductory biochemistry courses, so selecting a textbook for each course that maintains the distinctiveness of the course is a challenge. 1983–1984 was a particularly difficult year, and I took the opportunity to bend the ear of a publisher's representative. “You know so much about the problems with each book,” he said, “that you should be writing your own.” That prospect was sheer lunacy, but I remembered Ken van Holde, from our jointly taught class, as an outstanding speaker and writer. Also, as a biophysical chemist, he complements my interests, so I called on him. “I would have to be crazy to think about coauthoring a textbook,” he told me, “but yes, I am interested.” I was planning a sabbatical for 1984–1985 and thought (very näively) that I could work in the lab during the day and write at night. Our prospective editor, Diane Bowen of Benjamin Cummings, urged us to write an outline and prospectus and one sample chapter each. After I completed my sample chapter draft, I realized that writing thirteen or fourteen of these was going to be far more work than I was prepared for. But it was too late; reviewers of our prospectus liked it, and Ken was excited. Why did we even consider writing a textbook? I had always thought of myself as broad-based, and also I really wanted a book tailored to our one-year majors' course, in which I was teaching. Another factor was a wish to help define biochemistry as a discipline. In an era when biochemistry seems to be losing its identity vis-à-vis other molecular life sciences and when major universities have reorganized life sciences so that a biochemistry department no longer exists in name, I believe that biochemistry is a distinct discipline, although it intersects with all of the life sciences. I wanted to attempt to define that discipline. I returned from Stockholm to Corvallis in July 1985, having completed little more than that first sample chapter. Ken was in the same place, so we were really behind the eight ball. The one feature that helped me retain my sanity as we raced to keep on schedule was our purchase of a small A-frame cabin at the Oregon coast, a bit more than an hour's drive from Corvallis. On Friday evening, Kate and I would load my computer, papers, and books into the car, and I could work through the weekend with far more composure than I could at home. Ken did much of his writing at his cabin in the Cascade Mountains or at his summer home in Woods Hole. Textbook writing is a humbling experience. I learned rules of grammar that I had long forgotten. Our book was written in three drafts, and each chapter draft was read by four to eight reviewers, who pulled no punches. We were continually made aware that this is a business and not only must the science be good, but that it must be appropriate to the “market.” So we were given a lot of negative inputs to go along with the positives. Finally, sometime in 1988, the first draft was completed, and then we had to go through the process twice more. We had numerous arguments with