It seems like only yesterday.

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It Seems Like Only Yesterday Charles C. Richardson Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115; email: [email protected]

Annu. Rev. Biochem. 2015. 84:1–34

Keywords

The Annual Review of Biochemistry is online at biochem.annualreviews.org

DNA replication, bacteria, bacteriophage, replisome

This article’s doi: 10.1146/annurev-biochem-060614-033850

Abstract

c 2015 by Annual Reviews. Copyright All rights reserved

I spent my childhood and adolescence in North and South Carolina, attended Duke University, and then entered Duke Medical School. One year in the laboratory of George Schwert in the biochemistry department kindled my interest in biochemistry. After one year of residency on the medical service of Duke Hospital, chaired by Eugene Stead, I joined the group of Arthur Kornberg at Stanford Medical School as a postdoctoral fellow. Two years later I accepted a faculty position at Harvard Medical School, where I remain today. During these 50 years, together with an outstanding group of students, postdoctoral fellows, and collaborators, I have pursued studies on DNA replication. I have experienced the excitement of discovering a number of important enzymes in DNA replication that, in turn, triggered an interest in the dynamics of a replisome. My associations with industry have been stimulating and fostered new friendships. I could not have chosen a better career.

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Contents


GROWING UP IN THE SOUTH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . DUKE UNIVERSITY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . DUKE MEDICAL SCHOOL, DUKE HOSPITAL, AND EUGENE STEAD . . . . . . FIFTY YEARS OF MARRIAGE AND STILL COUNTING . . . . . . . . . . . . . . . . . . . . . . . PALO ALTO, STANFORD BIOCHEMISTRY, AND ARTHUR KORNBERG . . . . DECIDING ON A FACULTY POSITION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . THE EARLY YEARS AT HARVARD MEDICAL SCHOOL . . . . . . . . . . . . . . . . . . . . . . Glycosylated Bacteriophage T4 DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Polynucleotide Kinase and DNA Ligase: Last Days at the Bench . . . . . . . . . . . . . . . . . . Awareness of Other Replication Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Laboratory Milieu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Discontent and Career Decisions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ONSET OF STUDIES ON BACTERIOPHAGE T7 DNA REPLICATION . . . . . . . STANLEY TABOR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T7 DNA POLYMERASE AND DNA SEQUENCE ANALYSIS . . . . . . . . . . . . . . . . . . . CHAIRMAN OF THE DEPARTMENT OF BIOLOGICAL CHEMISTRY . . . . . . . THE T7 REPLISOME. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . COLLABORATIONS: THREE-DIMENSIONAL STRUCTURES AND SINGLE-MOLECULE ANALYSIS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Terminal Redundancy of Phage DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Crystal Structures of the T7 Replication Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Electron Microscopy of In Vitro Replicating DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Movement of the Replisome: Real-Time Single-Molecule Techniques . . . . . . . . . . . . Inhibitors of Prokaryotic DNA Primases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CONNECTIONS WITH INDUSTRY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hoffman–Roche . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . United States Biochemical Corporation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genetics Institute, Inc. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SERENDIPITY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Rediscovery of E. coli dGTPase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A Novel Nucleotide Kinase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T7 Gene 5.5 Protein, E. coli H-NS, and Transfer RNA Priming . . . . . . . . . . . . . . . . . . ANNUAL REVIEW OF BIOCHEMISTRY: A PLEASANT TASK . . . . . . . . . . . . . . . . . . . EPILOGUE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

3 4 4 6 7 8 8 9 9 11 11 11 12 14 14 15 16 17 17 17 17 18 18 18 18 19 20 20 20 21 21 21 22

“Hereby, too, I shall indulge the inclination so natural in old men, to be talking of themselves and their own past actions.” —Benjamin Franklin, Memoirs “Ah, but I was so much older then/I’m younger than that now.” —Bob Dylan, “My Back Pages”

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GROWING UP IN THE SOUTH WGTM, the call letters for the World’s Greatest Tobacco Market, broadcast at 1310 kHz in Wilson, North Carolina. I was born on May 7, 1935, in this modest town, where city-block-sized warehouses infused the late summer air with the aroma of freshly cured bright-leaf tobacco. Lying approximately 40 miles east of Raleigh, this region lures locals and visitors alike to its famous pork barbecue establishments. There are distinct differences in this Southern dish within a radius of only a few miles, a difference so great that I will drive a considerable distance to reach Parker’s BBQ on Highway 301 in Wilson. My father, Barney Clifton Richardson, was raised about 20 miles away near a small town, appropriately named Micro. His father, James Malachi Richardson, was a miller. His small farm lacked electricity and running water and was characteristic of the many rural areas of the South during that period when subsistence living was common. I remember weekend visits sleeping on featherbeds and catching the chickens to be cooked on the wood stove fired by my grandmother, Viola Maud Sellers. My own father was an accountant at a local automobile dealership, and my mother, Elizabeth Barefoot, was a housewife. I was an only child, no doubt in part due to the Great Depression with its financial difficulties. Childhood in Wilson was largely uneventful. My father, although he had no formal scientific training, fostered in me a keen interest in experimentation by supplying an endless array of mechanical parts from the auto service department. I recall at an early age trying to understand the water level in pipes with U-tubes. Then there was the Ford ignition coil that converted 12 volts to several thousand, sufficient to give quite a shock to unsuspecting victims. A car battery for which I had a generous supply of concentrated sulfuric acid powered this fascinating toy. I was determined to get a Gilbert chemistry set and sold garden seed from a company identified in a comic book to earn one. Quickly exhausting the sulfur, potassium nitrate, and charcoal for the manufacture of gunpowder, I went to the local pharmacy, where the benevolent pharmacist happily provided me with all I desired. Could one imagine that happening today? I have been impressed with the number of scientists who recall those days when chemistry sets were not restricted to reactions rewarded only with color changes. Fireworks and air rifles were ordered through the mail, with the former shipped by train. Growing up in the South in the heart of the Bible Belt implied Sunday school, church service on Sunday morning and evening, and Bible school on Sunday and Wednesday nights—not that I attended them all. Many counties were dry, in that no alcoholic beverages, aside from beer and wine, were sold. The South was segregated, and the only black adults I knew were maids, laborers, tenant farmers, or barbers. During my years of study at Duke University and Duke Medical School, there were still no black faculty or students. Public school was only a few blocks from my home, and from first grade on I walked to and from it every day. At the time, the professional careers attainable for women were nursing and teaching. As a consequence, I was fortunate to have had many dedicated teachers throughout my 12 years of public school who fostered the excitement of learning. Equally important was the discipline my mother instilled in me for laboring over homework and all else required for obtaining a good report card. When I was 11 we moved to Columbia, South Carolina. I entered the Columbia public school system in the sixth grade and in due course moved on to Dreher High School. Dreher High School was larger than any I would have attended in Wilson and, I suspect, a better school. Kary Mullis, who received the Nobel Prize in Chemistry for the polymerase chain reaction (PCR), attended Dreher High School. I wonder if we both had Mr. Wittenberg as our chemistry teacher; he focused on balancing equations and the gas laws, both of which served me well in college. I was studious and did exceptionally well in high school. Because I detested competitive team sports, I

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had considerably more time for academics than did many of my fellow students, and I was spared the physical limitations that frequently hamper aging athletes.

DUKE UNIVERSITY


My parents would have been pleased if I had become a physician or a pastor, the two pillars of a Southern community. The former appealed to me, as a solid science background was essential for admission to medical school and my favorite subject was science. I even took four years of Latin, another asset for medical school applicants. However, the greatest influence on my choice of colleges came from my cousin Henry Rogers, a pediatrician just starting practice after a stint in the Navy. Henry took me to visit Duke University. On campus he escorted me to the Dean of Admissions, who encouraged me to apply with an assurance that scholarships were available. I applied to Duke University and was accepted with a scholarship that paid my tuition of $350 per semester. That trip to Duke University dramatically influenced my career, and I have always been grateful to my cousin for devoting that day to me. The scholarship was important. I suspect my parents would have been considered financially poor, but I was never conscious of that circumstance nor of being stymied by unachievable goals. In high school I worked some afternoons and Saturdays in a local Winn-Dixie Supermarket, progressing from bag boy to checkout clerk to the produce department, and even to a stint in the meat department, where I was relegated to converting whole chickens to “cut-up” chickens. During the summers I worked 50-plus hours at $37.50 per week. I preferred to bag groceries and carry them to the customers’ cars; as a result, 25-cent tips rapidly accumulated. My parents drove me from Columbia to Duke University in Durham, North Carolina, in the fall of 1953. I spent my first night away from home in a monastic dormitory room, which I shared with two roommates. I majored in chemistry, one of the majors for premeds. Although advised to take only four courses, I chose five—math, English, French, chemistry, and biology—unaware of the time-consuming laboratory sections associated with the last two subjects. Consequently, only the evenings were available for studying. During those undergraduate years, there was no introduction to DNA. My biology textbook mentioned only that genes are chemical substances, most likely proteins, as only proteins are sufficiently complex to serve the function of the genetic material. In the summers I continued to work at the Winn-Dixie Supermarket and supplemented my income as an assistant in the math department by grading assignments.

DUKE MEDICAL SCHOOL, DUKE HOSPITAL, AND EUGENE STEAD I completed my third undergraduate year and entered Duke Medical School in 1956 without ever earning a bachelor’s degree. In the classical first-year medical school curriculum, gross anatomy occupied a full semester and left no foramen or fossa unexplored. I enjoyed my studies thoroughly and to this day can trace the branches of the trigeminal nerve and picture the papilla of Vater. Philip Handler, chairman of the Department of Biochemistry, was a formidable individual who went on to become President of the National Academy of Sciences and to receive the National Medal of Science, in part for his studies on the molecular basis of pellagra (1). Handler was a superb lecturer and gave the majority of the lectures using the recently published Principles of Biochemistry, which he coauthored with White, Smith, and Stetten—my introduction to this discipline (2). In the three years since my biology course, experiments by Avery et al. (3) implicating DNA as genetic material had been included in the textbook, but the structure of DNA had not. DNA polymerase was just being discovered in Arthur Kornberg’s laboratory at Washington University (4). During my second year of medical school, I learned of a new National Institutes of Health (NIH) program, the United States Public Health Service Post-Sophomore Research Fellowship, 4

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that provided a stipend to support one year of research. George W. Schwert, a professor in the Department of Biochemistry, offered me an opportunity to work in his laboratory, so I applied for and received the fellowship for the 1958–1959 academic year. George Schwert was a physical biochemist studying lactic dehydrogenase as a two-substrate enzyme (5). After a month collecting colostrum from pregnant women and assaying for lactic dehydrogenase in an attempt to screen for preeclampsia, I asked for a more challenging problem. I purified lactic dehydrogenase from dog heart and characterized it physically and enzymatically. The goal was to compare it with the beef heart enzyme characterized in the Schwert laboratory. I learned protein purification by preparing hydroxylapatite, as few chromatography resins were commercially available. The characterization included salting out, determination of electrophoretic mobility and the sedimentation constant, and a kinetic study. Particularly intriguing was the determination of mobility by means of a Tiselius apparatus, in which the protein was placed in a U-tube and the boundary of the protein as it moved through the electric field was recorded using Schlieren optics. The apparatus itself, with its long optical tube, occupied a full room. Gel electrophoresis lay in the future. Sedimentation analysis was at the forefront of molecular-weight determination. During my first run on the Model E Ultracentrifuge, the rotor fell into the chamber and all the components had to be replaced. I had most likely failed to tighten the connection of the rotor to the drive cable sufficiently. This was not an auspicious beginning, but I was never reprimanded, and I subsequently carried out many runs without incident. My year in the Department of Biochemistry provided an opportunity to take additional courses, including physical chemistry and advanced organic chemistry at Duke University and protein chemistry in the graduate school. This year in a lab was relaxing and fun. Once I felt comfortable, I introduced water-pistol fights, and I was surprised by an attack by George Schwert himself. I completed a thesis and received a BS degree in medicine to compensate for my premature departure from college. Jim Wyngaarden, an expert on purine metabolism (6), reviewed my thesis. Wyngaarden went on to become the Director of the National Institutes of Health and subsequently the Chairman of the Department of Medicine at Duke Hospital. That year sealed my determination for some research endeavor in my future career. Although my clinical experience was limited, I had decided to specialize in internal medicine. Consequently, I met with Eugene Stead, the Chairman of the Department of Medicine. He provided me with a proposal I had not anticipated: Return to medical school and complete the medical, pediatric, surgical, and obstetrics–gynecology rotations, but skip the senior medical and elective rotations and graduate with my class of 1960—essentially skipping a year of medical school. One of Stead’s many legendary quotes that may explain his generosity was, “A doctor doesn’t really need much knowledge, as he can look up most things.” This offer was dependent on my serving on Stead’s medical service. He may have reasoned that one week on his service would more than compensate for the courses I had skipped. He was correct: I passed the North Carolina Medical Boards, and entered the residency program at Duke Hospital in 1960. Medicine at Duke was an endurance test with total submersion into patient care. Eugene Stead was an extraordinary physician and teacher (7). Medicine today is influenced greatly by his innovative approaches, including his development of the position of physician’s assistant. At Duke, the interns lived in the hospital and were on call 24 hours a day, five days a week. Only the interns could write orders, draw blood, or start IVs. I cannot recall a night during my service on the public wards when I was not awakened for some pressing matter. To this day, I cringe at the sound of a telephone. Patients, particularly public patients, were very sick by the time they arrived on the wards. They could not afford the time to see a physician when they had a myocardial infarction, appearing only when they developed an arrhythmia or congestive heart failure. Unlike today, the www.annualreviews.org • It Seems Like Only Yesterday

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hospitals were judged on the number of autopsies; the numbers depended on the house staff ’s skill in persuading the relatives of the value of the procedure. I had the honor of winning the prize for the most autopsies: 20, if memory serves me. I like to believe that award was testament to my persuasive abilities and not to my incompetence. The result of this grueling year on the wards was an understanding of patients, their diseases, and their relationships. Stead’s remarks could on occasion be quite cutting: “If you can’t get your work done in 24 hours, you’d better work nights,” or “What this patient needs is a doctor” (8). One quickly grasps that with six patients being admitted you do not have time to do everything that you might otherwise wish, a lesson that has served me well over the years. I thoroughly enjoyed my first year of residency and the satisfaction of gaining a wealth of clinical knowledge. From interactions with patients from all walks of life, you learn that everyone does not think as you do. At the time, I actually intended to return to medicine and to complete my residency training. Although experimental science ultimately captured me, I have steadfastly maintained my license to practice in North Carolina. During my last year in medical school, I realized that I was certain to be drafted into the military during the Vietnam War. My plan was to perform alternative duty by joining the Public Health Service; people in this position were nicknamed the “Yellow Berets.” Hoping to continue my interest in protein chemistry, I drove to Washington, DC, in 1959 and visited the laboratory of Bill Harrington, only to learn that he had accepted a faculty position at Johns Hopkins University. I then decided to pursue a postdoctoral fellowship in any good laboratory, regardless of the military draft consequences. George Schwert mentioned that there was a young scientist at Stanford Medical School doing interesting work; I had never heard of Arthur Kornberg (9). On this advice and a concurrence from Phil Handler, I wrote a one-page letter to Kornberg sans curriculum vita. With letters of reference from Schwert and Handler, I received an acceptance letter. I applied for a Public Health Service fellowship and asked Arthur what information I should enter for the research plan. The entire Proposed Plan of Approach consisted of two sentences: “Detailed plans and techniques are available in Dr. Kornberg’s laboratory. I hope to learn them and participate in extending them as necessary for the pursuit of these investigations.” I received the fellowship. How delightful it would be to return to that Sputnik era.


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FIFTY YEARS OF MARRIAGE AND STILL COUNTING In the fall of 1960 I met Ute Ingrid Hanssum, a first-year graduate student in physiology, at Duke Medical Center. Our romance blossomed during one or two free nights per week and despite the fact that, due to extreme sleep deprivation, I drifted off to sleep at any opportunity. Ingrid had immigrated to Ithaca, New York, from Stuttgart, Germany, and had graduated from Cornell University. Before departing to Stanford together, we were married on July 29, 1961 in the Gothic Duke Chapel. Ingrid completed her PhD thesis at Stanford during my postdoctoral period, carried out a postdoctoral fellowship at Harvard, and became a faculty member in the Harvard School of Public Health Toxicology Department, a new department created by Armen H. Tashjian. During my early career, she proofread all of our manuscripts. Her English was better than that of any native speaker, a fact she attributed to her respect for the simple declarative sentence. After we arrived in Boston, our two sons, Thomas and Matthew, were born. Thomas has been editor of several fishing/boating magazines and a web magazine out of his home in Mattapoisett, Massachusetts, as well as host of a TV show. Matthew is a pediatric hematologist/oncologist at Bay State Medical Center in Springfield, Massachusetts, near his home in Granby. They are close enough to us for several visits each year, but sufficiently distant to preclude babysitting for our five grandchildren on a regular basis. 6

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PALO ALTO, STANFORD BIOCHEMISTRY, AND ARTHUR KORNBERG We arrived in Palo Alto in July 1961 after the cross-country honeymoon from Durham. After finding an apartment in Palo Alto, I reported to work. Arthur suggested that an initial project would be to improve the purification of DNA polymerase, the only known prokaryotic polymerase at the time. My first task was to master the assay. I was taken through the procedure by Arthur’s oldest son, Roger, then a high school student. Fortunately, I did not have to grow large quantities of Escherichia coli, as arrangements had been made with the Grain Processing Corporation of Muscatine, Iowa, to supply 100-pound batches of frozen cells. Using large Waring blenders, George Buggs prepared extracts. I persevered, and soon had a preparation that appeared homogeneous by chromatography and sedimentation analysis (10). How many pounds of E. coli did I process? Perhaps 2,000 pounds— 1 ton. This dedication to enzyme purification went far in obtaining Arthur’s favor, as he had little tolerance for those who wanted to get on to “something interesting.” The preparation of [α-32 P] deoxyribonucleoside 5 -triphosphates was a task expected of those of us using these essential components. E. coli was grown in low-phosphate medium with 100 mCi of [32 P]-inorganic phosphate. All radioactivity was taken up by the cells, which were suspended in a few milliliters of buffer, then washed and lysed; the lipids were extracted; and the DNA was hydrolyzed to mononucleotides. The four nucleotides were separated on an ion-exchange column, and each nucleotide was then enzymatically converted to the nucleoside triphosphate and once again fractionated. The entire process required three weeks of anxiety-ridden work for fear of having to restart the preparation. The dangers involved were likely not fully appreciated at the time. Naturally, I was not fazed when I needed [α-32 P] ATP of high specific activity a few years later at Harvard. Arthur’s laboratory group was relatively small. Upon my arrival, there were Vas Aposhian, a research fellow, and LeRoy Bertsch, a senior technician. John Josse, who had carried out the elegant nearest-neighbor analysis of DNA, was preparing to leave, ironically to join Bill Harrington’s group, where I had originally considered applying. Carl Schildkraut arrived, having completed his work in Paul Doty’s lab at Harvard. His many contributions on thermal melting profiles and the buoyant density of DNA were a marked contrast to my unfamiliarity with nucleic acids. John Jackson from Australia joined the lab later, as did Reiji and Tsuneko Okazaki. The Okazakis originally worked on thymidine kinase, but they too became infatuated with DNA. This small group met every morning in Arthur’s office and went over the raw data obtained the previous day. During the day, as I recorded numbers from the scintillation counter, I would frequently find Arthur peering over my shoulder. This curiosity was in no way intended to increase anxiety and productivity but merely reflected his keen interest, even in the recovery of an enzyme from a column. Yes, we worked very hard. Although the university campus was only a few minutes distant, I ventured there only once, to buy an attaché case for my first airplane trip to a meeting at Rutgers University. We were well on our way to the 10,000 hours needed for success in any endeavor (11). There was no better department for immersion into the world of nucleic acids. Bob Lehman was working on the deoxyribonucleases of E. coli, Paul Berg on RNA polymerase, Buzz Baldwin on the physical properties of DNA, Dale Kaiser and Dave Hogness on phage λDNA, and Ross Inman on electron microscopy (EM) of DNA molecules. The department was sufficiently small for daily communications, so seldom did a day pass without an awareness of new developments. Josh Lederberg’s Department of Genetics adjoined the Department of Biochemistry, and I developed collaborative efforts with Lederberg, A.T. (Gan) Ganesan, and Walter Bodmer in attempts to synthesize biologically active Bacillus subtilis DNA (12). In addition to these stimulating colleagues, a steady stream of visiting scientists flowed through the lab. H. Gobind Khorana visited frequently,

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and I recall evenings spent at a coffee house on University Avenue, where the latest experiments were discussed. Arthur had tried diligently but unsuccessfully to recruit Gobind to Stanford. Gobind was convinced that the downfall of Western civilization would start in California. He eventually went to MIT, where our contacts continued. Purifying an enzyme exposes interesting problems, and DNA polymerase was no exception. During chromatography of the purified enzyme, the recovery of activity was only 10% but with 100% recovery of protein. The addition of an adjacent fraction restored activity, leading to the discovery of E. coli exonuclease III (13, 14). Exonuclease III carried out a stepwise removal of nucleotides from the 3 terminus of the DNA and, if present, removed a 3 -phosphate as an initial step. Exonuclease III provided me with a reagent to show that DNA polymerase could repair partially degraded duplex DNA to restore full-length duplex DNA molecules (15). Bill Studier, a research fellow with Dale Kaiser, was studying the sedimentation properties of bacteriophage T7 DNA. Bill convinced me that this homogeneous DNA was far better than the salmon sperm DNA we routinely used. Indeed, the removal of nucleotides by exonuclease III and their replacement with DNA polymerase could be followed by EM. This product was easier to interpret than the complex structures obtained with duplex DNA (16). A second fringe benefit of polymerase purification was that it provided evidence that exonuclease II, identified by Bob Lehman, was physically associated with DNA polymerase (17). Arthur dismissed attempts to assign the two activities to a single protein, preferring to consider the exonuclease a pesky contaminant. He even wagered Buzz Baldwin a bottle of champagne, and only years later did he accept this enzymatic proofreading activity.


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DECIDING ON A FACULTY POSITION I could not have entered science at a better time. The NIH was pouring money into basic research, I had entered a field not yet represented at many institutions, and baby boomers had not saturated the market. I had no need to apply for faculty positions, but rather to decide which invitations to decline. Although the Bay Area was a delightful place to spend two years, we gravitated to positions on the East Coast. So, following a Federation Meeting in Atlantic City, I visited the Boston area, where I had been invited by the Biochemistry Department at Brandeis University, the Biology Department at Harvard College, and the Department of Biological Chemistry at Harvard Medical School. On three consecutive days I visited those departments and was offered positions at all three. I chose Harvard Medical School. The Biology Department at Harvard College was hard to decline, as Jim Watson was very persuasive. I visited Harvard Medical School on Saturday morning and met only with Elkan Blout and the chairman, Eugene Kennedy; I did not even present a seminar. The limited teaching obligations and a perception that I could pursue some clinical endeavor attracted me. I was not yet familiar with Harvard-affiliated hospitals, whose relationship to the school is different from that at Duke. I was offered the position of Instructor, a title I was told was identical to Assistant Professor at other institutions. “So let’s just make it an Assistant Professor position,” I suggested. I suspect the other Instructors were unaware of what triggered their promotions.

THE EARLY YEARS AT HARVARD MEDICAL SCHOOL We arrived in Boston in February 1964 to find ourselves in a snowstorm without snow tires. My single laboratory room had been recently renovated by Chris Anfinson, who had been recruited from NIH but had decided that Harvard Medical School was not his cup of tea. His departure showed that the NIH slogan “NIH, a Pathway to Academia” was actually a reversible pathway. 8

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I was fortunate in that he left behind a spectrophotometer and a preparative ultracentrifuge. I had applied for an NIH grant, and funds were available upon my arrival. Start-up funds were not offered, nor were they needed. I set to work on an initial project. After nine months of gestation, we purchased a house in Chestnut Hill, one of the villages of the Town of Newton. It was near three subway lines and only 60 minutes by foot or 20 minutes by bicycle to Harvard Medical School. To this day I bicycle to work, except when snow covers the road. At $32,500, the house was beyond our financial means, but we had saved the down payment from our stipends at Stanford.


Glycosylated Bacteriophage T4 DNA Before leaving Stanford, I had noticed that exonuclease III degraded bacteriophage T4 DNA at a very slow rate. T4 DNA, like the DNA from the other T-even phage, contains hydroxymethylcytosine in place of cytosine, and this residue is glycosylated to varying patterns, depending on the T-even phage. The glycosylation is part of a restriction–modification system unique to T-even phage (18). In my first experiments at Harvard, I found that E. coli exonucleases II and III had difficulty degrading glycosylated DNA but not DNA containing nonglycosylated hydroxymethylcytosine (19). Bob Lehman (20) had previously shown that exonuclease I degraded glycosylated DNA. However, extracts of bacteria that allowed for the growth of T-even phage containing nonglycosylated DNA contained normal amounts of these enzymes. These studies led to a collaboration with Charles Thomas at John Hopkins University; in 1967 we showed (21) that T2 DNA had a terminal redundancy, identified by circle formation after removal of several hundred nucleotides by exonuclease III. Only by using nonglycosylated T2 DNA was this experiment feasible. A few years later, Charles Thomas joined the department and brought with him a breath of fresh air. Later, in 1971, Roger Fleischman, an MD–PhD student, showed that E. coli cells made permeable with toluene could incorporate hydroxymethylcytosine into their DNA, an event that halted DNA synthesis in strains that restricted phage with nonglycosylated DNA (22). Roger and Judy Campbell, also a graduate student, reproduced this phenomenon in vitro, but the activity proved elusive (23).

Polynucleotide Kinase and DNA Ligase: Last Days at the Bench My major initial goal was to identify an enzyme that would covalently join DNA molecules. It was becoming clear that several processes involved the joining of DNA molecules. Recombination studies showed that the breakage and joining of DNA, phage λDNA–formed circles in E. coli, and discontinuous synthesis with subsequent joining of the fragments (24) could solve replication problems. By analogy to the polymerization of nucleotides by DNA polymerase, I thought it not unreasonable that the 5 terminus of a polynucleotide could be activated by a triphosphate so that the 3 -hydroxyl group of another polynucleotide could catalyze a nucleophilic attack to produce a phosphodiester bond. With [γ-32 P] ATP in hand, I surveyed extracts of E. coli– and T4 phage– infected cells for their ability to phosphorylate the 5 termini of oligonucleotides prepared by treatment of DNA with pancreatic DNase or micrococcal nuclease. Extracts of T4 phage–infected cells demonstrated robust activity for the phosphorylation of 5 -hydroxyl-terminated oligonucleotides (25). Novogrodsky and Hurwitz also described this enzyme, polynucleotide kinase, in extracts of T2 phage–infected cells (26). Although polynucleotide kinase was not involved in the activation of the 5 termini of DNA, it proved to be a valuable reagent. I labeled the 5 termini of T7 DNA with 32 P by using polynucleotide kinase. By this end-group labeling I calculated the length of a strand of T7 DNA as www.annualreviews.org • It Seems Like Only Yesterday

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being 40,000 nt (27). Eventually, DNA sequence analysis of the T7 genome revealed a length of 39,936 nt, an error of only 0.2% (28)! I also isolated the 5 -labeled nucleotides after nuclease digestion and found that one strand was terminated by A and the other by T. I extended this crude DNA sequencing to determine the 5 dinucleotides by taking advantage of the inability of E. coli exonuclease I to cleave a dinucleotide (29). These two bases represented the longest sequence of a DNA molecule at the time, and each base warranted a paper! I also showed that shearing of duplex DNA cleaves the phosphodiester linker to give 5 -phosphoryl and 3 hydroxyl groups in a ratio of 20 to 1, reflecting the relative stability of primary and secondary alcohols. During this period David Hinkle, an undergraduate at Harvard College, pursued his honors thesis by determining the specificity of phosphodiesterase of Crotalus adamanteus. David returned to my lab as a research fellow after obtaining a PhD with Mike Chamberlin at the University of California, Berkeley. Although not involved in the covalent joining of DNA, polynucleotide kinase proved valuable in identifying this activity. A likely DNA intermediate, just prior to joining, would be a nick in duplex DNA in which the 5 -phosphoryl termini and the 3 -hydroxyl termini are juxtapositioned. Therefore, a DNA molecule containing a nick with a 32 P-labeled phosphate would be the ideal substrate for measuring joining by the protection of the [32 P]-phosphate from phosphatase. Fortunately, to assist in these efforts, my first postdoctoral fellow, Bernie Weiss, arrived in 1965, after completing his military service at Walter Reed Army Institute of Research. Bernie had carried out his residency in medicine prior to military service, and his USPHS Special Fellowship was considerably more than my $11,000 faculty salary. Bernie set out to label the phosphates at nicks generated by pancreatic DNase—not an easy task. Those phosphates first had to be removed prior to replacement with [32 P]-phosphates. Such internally located phosphates are resistant to phosphatase activity, requiring an elevated temperature of 65◦ C (30–32). Likewise, the kinase acts at these internal positions only at elevated temperature. These studies fostered separate projects in which Yukito Masamune examined the action of exonuclease and polymerase at nicks (33, 34) and Immo Scheffler used DNA covalently linked to Ficoll, a polymer soluble in aqueous solution (35). Bernie succeeded, and his substrate led to the identification of a joining activity in extracts of T4 phage–infected cells that we designated DNA ligase (36, 37). Once purified (38), the reaction mechanism became a fascinating study. Bernie Weiss; George Fareed, a medical student; Elaine Wilt, a technician awaiting entry into medical school; and Yukito Masamune isolated the protein and DNA intermediates (39–41). Within a short time frame, the labs of Marty Gellert, Bob Lehman, Jerry Hurwitz, and Arthur Kornberg independently described DNA ligases by using novel assays (42–45). George Fareed identified the structural gene for T4 ligase (46), and Yukito Masamune examined the fate of DNA within phage-infected cells lacking the T4 ligase (47). The availability of DNA ligase fostered a number of studies. Ted Live, my first graduate student, used end-group labeling and DNA ligase to determine the identity of the terminal nucleotides of the strands of phage λDNA (30, 38). Alain Jacquemin-Sablon showed that the single-strand breaks in T5 DNA consisted of nicks located in only one of the two strands (48). We unsuccessfully attempted to identify an enzyme that introduced these nicks, but Gerald Frenkel, a graduate student, did isolate a phage T5 5 -exonuclease (49, 50), an enzyme described earlier by Paul and Lehman (51). Polynucleotide kinase and DNA ligase were valuable enzymes in studies on nucleic acids. DNA ligase played a major role in covalently linking small oligonucleotides to form longer polymers (52). Gobind Khorana visited the lab on several occasions to obtain enzymes or to assist in their purification.


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Awareness of Other Replication Proteins The discoveries of DNA ligase and Okazaki fragments provided an explanation for the synthesis of the lagging strand. There was increased awareness of the involvement of other proteins arising from the isolation of E. coli mutants defective in DNA replication (53). And then there was the E. coli mutant isolated by Paula De Lucia and John Cairns in 1969 (54) that had no detectable DNA polymerase activity in extracts yet grew normally. Robb Moses in my lab used toluenetreated cells, permeable to nucleoside triphosphates, to examine replication deficient strains as well as the Cairns mutant and showed there was a requirement for ATP (55), suggesting energydependent steps. In parallel studies, Robb identified DNA polymerase II in extracts of the Cairns mutant (56–58). Judy Campbell assayed extracts of mutagenized cells and discovered that the thirty-fifth strain lacked this activity (59, 60); she was more fortunate than Paula De Lucia, who assayed 3,478 colonies before finding the polymerase I mutant. Tom Kornberg and Malcolm Gefter also identified DNA polymerase II and its gene (61–63). They proceeded to identify the replicative DNA polymerase, DNA polymerase III (63, 64). It was an exciting period in DNA replication.

Laboratory Milieu Experimental science has always been fun for me, and I have assumed that is true for others. Therefore, I have never applied pressure for prolonged hours at the bench or for specified periods. Rather, I have attempted to foster an environment that does not distract from the excitement of research. In the early days, when there were few restaurants in the area, we had daily lunches in the hallway. Usually we had hamburgers on Monday, hot dogs on Tuesday, tuna sandwiches on Wednesday, and chili on Thursday, and the big lunch on Friday consisted of roast chicken or a beef or pork roast. All were cooked on a rotisserie, also in the hall, that infused the building with wonderful aromas. As the lab grew and the number of local eateries increased, along with the institutional regulatory bureaucracy, the lunches were reduced to Mondays and Fridays, with one lab member responsible for each. Many of the spouses were delighted that their partners actually learned to cook. I believe that most considered the lab a pleasant place to spend their time away from home. As more people arrived and manuscripts proliferated, it became apparent that a secretary would be an asset, so in 1967 I hired my first and only secretary, Bess Swartz. Bess assisted me over her 35-year tenure in establishing the lab, caring for students and fellows, and for a 10-year period serving as administrative assistant when I was chairman of the department. Bess was probably the fastest typist at Harvard University, an attribute that you cannot appreciate unless you started your career before the computer era. Bess Swartz retired in 2001 at the age of 84. I have had few research assistants. Ann Dolan (Thompson) joined the lab shortly after I arrived and was responsible for routine enzyme assays. In 1981 Benjamin Beauchamp joined the group and has remained my sole research assistant ever since. Ben initially played a major role in the experimental work and worked on several projects such as the dGTPase of E. coli. In more recent years, Ben has devoted more time to laboratory maintenance and the safety regulations that seem to multiply like rabbits.

Discontent and Career Decisions The transition from Stanford to Harvard Medical School was not easy. The Stanford department, only recently founded, had all the modern facilities and equipment. Arthur had recruited the entire www.annualreviews.org • It Seems Like Only Yesterday

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faculty, and everyone pursued nucleic acid problems. If they were not already doing so when they arrived, they were quickly converted. Thus, there were many opportunities for collaboration and helpful advice from adjoining laboratories. Research supplies were obtained from the stockroom, having being purchased by shared funds. The weather was also pleasant. The department at Harvard was some 200 years older. The buildings were in ill repair, with no central air conditioning and poor heating in winter. Water pipes burst frequently. Stairs provided the quickest access to each floor, although the cagelike elevator provided panoramic views as it slowly but faithfully ascended. Upon my arrival in 1964, there was neither ice nor Xerox machines. The one autoclave resided in the laboratory of Ed Lin, and sterilization of media necessitated one’s presence to continuously regulate the pressure. The purchase of a large pressure cooker freed up time for other activities. The faculty in the department was diversified. Don Comb, pursuing studies on 5S RNA, was the only other nucleic acid biochemist in the department. Don left academia and opened New England Biolabs in his basement. I recall providing him with T4 phage and protocols to prepare polynucleotide kinase and DNA ligase in those days, prior to the Bayh–Dole Act. My isolation was breached by the biochemists at Harvard College across the river. Jim Watson invited me to attend their evening group meetings, so I could keep up with the rapidly evolving field. In those years the Committee on Biochemistry at Harvard College far outshone the department at Harvard Medical School. When I accepted the position at Harvard, I did so with the realization that a junior faculty member was unlikely to achieve tenure. I solicited an opinion only to be told it was premature to consider such a promotion. The Biochemistry Department at the University of California, Berkeley, offered me a tenured position, and after a visit, I accepted. I resigned from the department at Harvard and prepared to put my house on the market. Some months later, Ronald Reagan launched his political career by vowing “to clean up the mess at Berkeley,” which eventually culminated in the riots of 1969. Hearing of disruptions in research from my friends at Berkeley, I made a very difficult decision: I withdrew my acceptance. My obvious intention to depart from Harvard Medical School oiled some gears, as within a year I was promoted to tenure with the honor of being the youngest to achieve this distinction, at the age of 32. E.D. Churchill (65), the famous surgeon, was the previous holder of this distinction, and I believe Jon Beckwith eventually held the record. An additional issue was the need for medical doctors in the armed forces during the Vietnam War. I was continually submitting petitions to the local Selective Service Board in Columbia, South Carolina. Finally, in 1965 I received a notice to report for duty, requiring me to bring only an overnight change of clothes. A panicked call to the office in Columbia brought me into contact with the clerk of the board, who was delighted to hear from “the only one of her boys who had not yet been drafted.” After assuring me that there was no alternative, she provided me with the number of Colonel Collins at nearby Fort Jackson. My conversation, using my best Southern accent, with Colonel Collins resulted in a telegram informing me not to report to duty.


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ONSET OF STUDIES ON BACTERIOPHAGE T7 DNA REPLICATION We began studies on E. coli DNA replication. Dennis Livingston carried out his thesis work on DNA polymerase III and its associated exonuclease (66, 67). Jack Chase identified E. coli exonuclease VII, whose novel specificity made it a valuable addition to nucleic acid reagents (68–71). Hiroaki Shizuya developed an in vitro replication system for phage λDNA, which depended on E. coli replication proteins (72). However, the competition in this field made this area less attractive, so in the early 1970s Melvin Center and Pasquale Grippo, postdoctoral fellows, initiated studies 12

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on T7 DNA replication. After leaving Stanford, Bill Studier undertook studies at Brookhaven National Laboratory on bacteriophage T7. He identified the 20 essential genes, and his genetic studies provided insight into their in vivo roles (73, 74). On several occasions Bill had encouraged me to examine DNA replication in such a defined system. Melvin characterized the gene 3 endonuclease (75–77), and Pasquale identified the gene 5 product as a DNA polymerase (78). Yukito Masamune identified the DNA ligase gene and showed that the host enzyme could replace it (47, 79). Gene LeClerc showed that the gene 2 protein was an inhibitor of E. coli RNA polymerase, a requirement for DNA packaging (80). In a collaborative effort with Bill Studier, Judy Campbell showed that fragments of T7 DNA cloned into E. coli could provide functions for infecting phage (81). More importantly, these fragments could recombine with the DNA of infecting phages, thus integrating the physical and genetic maps. T7 DNA polymerase has been a steady source of surprises. Mike Chamberlin isolated mutants of E. coli that could not support the growth of T7 phage (82). One of these mutants, E. coli tsnC, exhibited no phage DNA synthesis after infection with T7. Paul Modrich found that extracts of the T7-infected tsnC mutants were devoid of T7 DNA polymerase activity (83). Purification of a component that restored activity yielded a 12-kDa heat-stable protein (84). At a Cold Spring Harbor meeting, Neal Brown suggested that thioredoxin was a likely candidate, and when David Mark added it to the gene 5 protein, activity was restored (85). Katsuji Hori, on sabbatical from Kyushu University, purified the protein from E. coli lacking thioredoxin and found that it had 1–2% of the activity of the gene 5 protein–thioredoxin complex (86). However, we attributed this low activity to contaminating thioredoxin. It was not until later that we realized the gene 5 protein itself was a DNA polymerase with low processivity. Katsuji also showed that the gene 5 protein had an active 3 –5 exonuclease activity (87). David Mark mapped the gene for thioredoxin and showed that E. coli lacking thioredoxin had no phenotype except for the inability to support T7 growth (88). Other labs, in particular Arne Holmgren’s, set out to identify the protein that provided reducing power for ribonucleotide reductase, previously thought to be dependent on thioredoxin (89). Years later, Jaya Kumar in our lab published the E. coli thioredoxin proteasome, identifying the interactions of this component of all organisms (90). Having identified the DNA polymerase and the DNA ligase of T7, we could only speculate on the role of other proteins. As did other groups, we turned to establishing an in vitro T7 DNA replication system. David Hinkle in my lab (91) and Rolf Knippers and colleagues in Germany (92) developed replication systems that revealed a requirement for the product of gene 4 as well as the gene 5 DNA polymerase. Warren Masker showed that some of the molecules synthesized in the in vitro system were intact genomes with biological activity (93, 94). David Hinkle then purified the gene 4 protein by using a complementation assay (95). Richard Kolodner showed that it allowed T7 DNA polymerase to copy duplex DNA, the first of the DNA replication helicases to be described (96–98). Later, Steve Matson studied the nucleotide and DNA specificity (99–102). Not only was the gene 4 protein a helicase but, as Eberhardt Scherzinger and colleagues (103, 104) in Berlin found, it was also a DNA primase, again the first of this class of enzymes to be identified. Lou Romano characterized the primase reaction (105, 106), and in 1981 Stan Tabor, a graduate student at the time, identified the primase recognition sequence in DNA (107). The gene 4 protein exists in two molecular-weight forms due to the presence of an internal initiation codon within gene 4. Julie Bernstein showed that the smaller form lacks the 63 N-terminal residues of the full-length protein and is devoid of template-directed primer synthesis because it lacks the Cys4 zinc motif (108–110). Hiroaki Nakai initiated studies on the multiple roles of the gene 4 protein in leading- and lagging-strand synthesis and demonstrated a role for the T7 single-stranded DNA (ssDNA)–binding protein (111–114). www.annualreviews.org • It Seems Like Only Yesterday

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Fuju Tamanoi (115) identified the primary origin of T7 DNA replication by deletion mapping, and Haruo Saito and Stan Tabor sequenced this region (116). The primary origin contained two T7 RNA polymerase promoters preceding a 61-bp AT-rich site. Lou Romano and Fuju Tamanoi were able to initiate DNA synthesis in vitro on a plasmid into which this origin had been cloned, provided that T7 RNA polymerase was present (117). Carl Fuller showed that DNA synthesis was initiated by extension of RNA transcripts (118, 119). DNA synthesis was unidirectional unless the T7 ssDNA–binding protein was present. Sam Rabkin showed that plasmids containing a T7 replication origin could be replicated by the T7 replication proteins in vivo, providing a way to identify the secondary origins that function when the primary origin is deleted (120, 121). During this period, Rick Ikeda used a proteolytically nicked RNA polymerase to examine its interaction with its promoter (122–124). T7 replicates as a concatemer that is processed into genome size as it is packaged into the phage capsid. Yeon-Bo Chang, who had worked as a graduate student on this process in David Hinkle’s laboratory, pursued this problem as a research fellow in my lab. John White, a graduate student, approached the problem from a more biochemical route, purifying the products of genes 18 and 19 and examining the processing of concatemers (125–127).


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STANLEY TABOR I would be remiss not to devote a special paragraph to Stan Tabor, who joined my lab in 1977 as a graduate student, having earned his BS and MS at Stanford University. Stan initiated work on the T7 DNA primase and mastered the new Maxam–Gilbert DNA sequencing technique to identify the sites on φX174 DNA, where primers were synthesized and extended by T7 DNA polymerase (107). As if this contribution were not sufficient for his thesis, Stan proceeded to develop a T7 RNA polymerase–promoter system for gene expression (128). With overproduced T7 DNA polymerase, Stan showed that the enhancement of activity by thioredoxin reflects an increase in processivity (129, 130). He found that selective oxidative damage to the enzyme by ferrous iron specifically inactivates the exonuclease activity, thus rendering it suitable for DNA sequence analysis (131, 132). I have been fortunate to have Stan as a student, as a research fellow, as an independent member of the lab, and as a friend over the past 37 years. I hope others recognize his contributions to the extent that I do.

T7 DNA POLYMERASE AND DNA SEQUENCE ANALYSIS Stan Tabor and Hans Huber found that the gene 5 protein itself did have polymerase activity and that thioredoxin stimulated DNA synthesis by increasing its affinity to the primer template (130). Mutationally altered proteins, obtained from Marjorie Russel and Peter Model, showed that the reducing potential of thioredoxin was not important in this role (133). Jeff Himawan, a graduate student, isolated phage T7 mutants that could grow on E. coli expressing one of these mutant thioredoxins, which could not support the growth of wild-type T7 (134, 135). Some of the suppressor residues resided within the gene 5 protein, and these residues provided contact points for the two proteins. Xiao-Ming Yang also showed that amino acid changes within this region altered its binding to thioredoxin (136). A comparison between gene 5 protein and the Klenow fragment of E. coli DNA polymerase I reveals a novel 76–amino acid sequence in the thumb domain, and these altered residues reside in this segment. Ella Bedford and Stan Tabor inserted this segment into the Klenow fragment, and indeed, it bound thioredoxin and the processivity increased (137). When David Hinkle established his research group at the University of Rochester, he made the puzzling observation that their T7 DNA polymerase could not initiate synthesis on nicked DNA 14

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in the presence of DNA helicase, in contrast to the result obtained in Boston (138). Stratling and Knippers had found a similar result (139). One difference in the two polymerases was the presence or absence of ethylenediaminetetraacetic acid (EDTA) during purification. Mike Engler and Bob Lechner in my group found that the presence of EDTA during purification yielded a DNA polymerase with high levels of exonuclease activity, whereas that purified in its absence had low levels (140). In the absence of exonuclease activity, T7 DNA polymerase catalyzed stranddisplacement synthesis at a nick, thus creating a ssDNA to which the helicase could bind (141, 142). Stan Tabor later demonstrated that T7 DNA polymerase could be converted to the form lacking exonuclease by Fenton chemistry (131). T7 DNA polymerase–thioredoxin had the potential for use in DNA sequence analysis. It is highly processive, and it does not discriminate against the chain-terminating dideoxynucleotides. However, the high exonuclease activity precluded its use in DNA sequencing. Stan Tabor took advantage of the exonuclease-free enzyme to show that the modified enzyme produced good sequencing results with sharp bands of equal intensity (132). Stan also examined the effect of manganese and the detrimental effect of a buildup of pyrophosphate (143–145). He subsequently eliminated the chemical modification by isolating genetically modified polymerases lacking exonuclease activity (146). How does T7 DNA polymerase incorporate dideoxynucleotides so efficiently? Other members of this family, such as E. coli DNA polymerase I and Taq DNA polymerase, discriminate several hundredfold. Stan found that a single hydroxyl group in T7 DNA polymerase is the determinant (147). Substitute phenylalanine for tyrosine-526, and the enzyme discriminates against dideoxynucleotides to the same extent as does DNA polymerase I containing phenylalanine at this position. Replace the phenylalanine in E. coli DNA polymerase I or Taq DNA polymerase with tyrosine, and the enzyme no longer discriminates.

CHAIRMAN OF THE DEPARTMENT OF BIOLOGICAL CHEMISTRY In 1978, I accepted the chairmanship of our department. The Chair of the Department of Biological Chemistry at Harvard Medical School, for many years, had a permanent incumbent, but Eugene Kennedy resigned after a few years, and by default the position rotated from one professor to another. Daniel C. Tosteson was appointed dean in 1977 and asked me to assume the chairmanship without a specified length of time. His enthusiasm for strengthening the basic science departments and my dissatisfaction with the previous departmental organization led me to accept his offer. My trepidations were relieved by knowing that the position was no more permanent than my own decision or that of the dean. Sandy Mulcahy, my former research assistant, had just received his doctoral degree from the Harvard School of Public Health and accepted my invitation to become the scientific administrator. Upon his departure several years later to join the faculty at Suffolk University, I persuaded Alex Nussbaum, who had moved from Hoffman–Roche to the Boston Biomedical Research Foundation, to assume this position. Alex also established a facility for the synthesis of oligoribonucleotides. Those nine years passed quickly. I found the search for new faculty members for the department gratifying, and I tried to remove the stigma of the lack of tenure appointments from within. However, I did not find the chairmanship as absorbing as my own research and at times resented the sacrifice of time and effort that come with the responsibility. In 1988 we were searching for a chairman for the Department of Pharmacology and identified Chris Walsh at MIT as the leading candidate. Dan Tosteson reluctantly informed me that Chris would consider chairmanship only of a combined Department of Biological Chemistry and Molecular Pharmacology. My heart leapt with glee. www.annualreviews.org • It Seems Like Only Yesterday

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THE T7 REPLISOME


Up to this point, I have discussed research interests in considerable detail. Due to limitations of space, I present the more recent work of the past 25 years with exponentially decreasing detail as the years advance. This strategy is not intended to deflect from the exciting work produced in the laboratory when I could only make suggestions and marvel at the results. These studies and the proper credits are well recorded in not-too-distant reviews (148–151). The outstanding individuals who participated in these studies and are mentioned only briefly elsewhere include Barak Akabayov (143, 152–158), Nathalie Andraos (159), Khandan Baradaran (160, 161), Bruno Canard (162–164), Kajal Chowdhury (162, 165, 166), Erica Chiu (167), Don Crampton (168–173), Zeger Debyser (174, 175), David Frick (148, 160, 176–179), Sharmistha Ghosh (180–183), Shenyuan Guo (168, 184, 185), Samir Hamdan (149, 180, 181, 186–197), Zhengguo He (198, 199), Alfredo Hernandez (200), Anna Hine (201, 202), Edel Hyland (203), Don Johnson (157, 168, 187, 190, 204, 205), Young-Tae Kim (206–209), Robin Kremsdorf (210), Arek Kulczyk (143, 153, 155, 183, 195, 211– 216), Takahiro Kusakabe (161, 202, 217–220), Daochun Kong (221–224), Jaya Kumar (90, 167, 176, 210, 225), Joon-soo Lee (161, 178, 220, 226, 227), Joseph Lee, Seung-Joo Lee (143, 150–152, 154–157, 166, 189, 190, 192–194, 196, 215, 228–240), Qingyun Liu (241), Boriana Marintcheva (182, 189, 192, 194, 242–245), Lynn Mendelman (246–250), Hitoshi Mitsunobu (251), Sourav Mukherjee (169, 172), Sandy Mulcahy (56, 227), Jill Myers (252, 253), Steve Notarnicola (227, 248, 249, 254, 255), Udi Qimron (170, 187, 195, 196, 228, 243, 244, 256), Lisa Rezende (198, 203, 256– 258), John Rush (259), Ajit Satapathy (171, 172, 183, 260), Jim Stattel (261), Masateru Takahashi (182, 188, 190), Akira Taketo, Ngoc Tran (157, 214, 234, 236, 256, 262), Timothy Tseng (179), Huidong Zhang (150, 215, 235, 236), and Bin Zhu (154, 156, 193, 200, 215, 236–240, 263). During the past 15 years I have been fortunate to have Seung-Joo Lee, a research associate, as a member of my group. Seung-Joo has carried out many elegant studies on T7 DNA primase. In addition, he collaborated with others in the lab and introduced both students and research fellows to the T7 DNA replication field. The 1980s through the early 2000s were concerned with the individual proteins involved in T7 DNA replication. During this phase, genetically altered proteins provided insight into essential functions and the assignment of function to various motifs. It became clear that interactions among these proteins are critical to the coordination of the events that occur during movement of the replication fork. The association of these proteins into a replisome secures these interactions. A functional T7 replisome consists of only four proteins: T7 DNA polymerase, E. coli thioredoxin, T7 helicase–primase, and T7 ssDNA–binding protein. However, each has usurped properties found in the accessory proteins of more complex systems. Consequently, considerable time has been spent in unraveling the reactions mediated by these four proteins. Studies of the DNA polymerase–thioredoxin complex focused on the proofreading exonuclease, on pyrophosphorolysis, and on processivity. Studies of the T7 helicase revealed properties common to all the hexameric helicases. The mechanism of oligomerization, nucleotide specificity, the arginine finger, interactions between the individual subunits, and the ability to promote homologous strand transfer are each a story in itself. The primase has been difficult to understand in molecular terms. The ability of the primase to recognize DNA sequences is a complex process involving an N-terminal zinc motif, the RNA polymerase domain, and the linker connecting the two. An interaction of the primase with the helicase may play a role in coordination of leading- and lagging-strand synthesis. In his initial studies, Bill Studier missed one essential T7 gene. In the absence of the gene 2.5 ssDNA-binding protein, there is no T7 DNA synthesis. This protein, like the T7 helicase, has an acidic C-terminal tail that binds to T7 DNA polymerase. Its interaction with the polymerase is essential for loading of the polymerase–helicase onto a nick in DNA. The protein, unlike other

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ssDNA-binding proteins, mediates homologous base pairing and dramatically increases the rate of annealing of complementary strands. For the past 15 or so years, research has focused on the interactions that coordinate the movement of the replisome. Zeger Debyser observed coordinated leading- and lagging-strand synthesis (174). In collaboration with Jack Griffith at the University of North Carolina at Chapel Hill, Kyusung Park observed the long-sought replication loops on the lagging stand (175). Stan Tabor suggested using a small chemically synthesized DNA minicircle, and Joon-soo Lee constructed such a molecule. With the minicircle of fewer than 100 nt, coordinated leading- and lagging-strand synthesis was observed (220, 226). Most importantly, most of the replicating molecules were found by Paul Chastain in Jack Griffith’s lab to have lagging-strand replication loops whose size accommodates the nascent Okazaki fragment. The dynamic state of the replisome poses problems in obtaining stable structures for structural determination. Crystal structures of subassemblies of the replisome have thus far been unsuccessful. As a result, we have resorted to small-angle X-ray scattering (SAXS) and cryo-EM to circumvent this obstacle.

COLLABORATIONS: THREE-DIMENSIONAL STRUCTURES AND SINGLE-MOLECULE ANALYSIS Terminal Redundancy of Phage DNA I have been fortunate in having outstanding collaborators. My first collaboration was with Charles A. Thomas, Jr., at John Hopkins University. That collaboration led to the demonstration that the sequences at each end of phage T4 DNA were redundant, as predicted by the genetic map (21). The studies were extended to phages T3 and T7 (264). Shortly thereafter Charles Thomas joined my department at Harvard Medical School, and our close contacts continued.

Crystal Structures of the T7 Replication Proteins In 1993 Tom Ellenberger joined the department. Our labs were located across the hall from each other. To my good fortune, Tom elected to determine the structures of the proteins of the T7 replisome. Stan Tabor had formed a stable complex of DNA polymerase–thioredoxin bound to a primer template in a polymerization mode. Sylvie Doublié, a research fellow in Tom’s lab, generated crystals for diffraction, and shortly thereafter we had a beautiful 2.2-A˚ structure (265). Soon to follow were the structures of the helicase and the primase (185, 266), the fulllength gene 4 protein (267), and the gene 2.5 protein (261). These structures were determined by Michael Sawaya, Masato Kato, Eric Toth, and Tom Hollis, all in the Ellenberger lab. The structures of these proteins elucidated many of the phenomena we had reported and suggested future approaches.

Electron Microscopy of In Vitro Replicating DNA My first introduction to EM came from studies on E. coli exonuclease III in Arthur Kornberg’s laboratory, where Ross Inman observed the processive shortening of T7 DNA by exonuclease III and its restoration by DNA polymerase (15). During my collaboration with Charles Thomas in 1966, Lorne MacHattie used EM to observe the formation circles after exposure of the terminal redundancy by exonuclease III (21). Early in our studies on T7 DNA replication, I was fortunate to have Jack Griffith as a collaborator, not only for his many contributions but also as a personal friend. One highlight was a scientific meeting in Alaska, his native state, where we met just outside www.annualreviews.org • It Seems Like Only Yesterday

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Denali National Park. As described above, his electron micrographs of the products of DNA replication in vitro revealed the first definitive existence of the replication loops proposed years earlier by Bruce Alberts and colleagues (268).

Movement of the Replisome: Real-Time Single-Molecule Techniques


In 2004 Antoine van Oijen joined the department. We had collaborated with Antoine when he was a research fellow in Sunney Xie’s group at Harvard College, where he observed the hydrolysis of DNA by λ exonuclease (173). At Harvard Medical School, Antoine used single-molecule techniques to examine DNA synthesis mediated by the T7 proteins. J.B. Lee determined the rate of movement of the polymerase and helicase and showed that primer synthesis temporarily halts leading-strand synthesis (191). C. Etson used these techniques to show that thioredoxin, the processivity factor, suppresses microscopic hopping by the polymerase (186). Sam Hamdan, a research fellow in my lab, joined Antoine’s group and showed that leading- and lagging-strand synthesis was coordinated with the formation and resolution of replication loops (188). The exchange of DNA polymerases in solution with those at the replication fork was observed directly by Joe Loparo and Arek Kulczyk (213), and Sam showed that it involved interactions of the C-terminal tail of the helicase with the polymerase (187).

Inhibitors of Prokaryotic DNA Primases Prokaryotic DNA primases are promising candidates for small-molecule inhibitors of microbial growth. The primases are essential to the cell, primases from different organisms exhibit different sequence specificities, and the prokaryotic primases differ significantly from eukaryotic primases (148). A major problem in screening libraries of small molecules is the lack of a rapid assay. Therefore, Barak Akabayov undertook a collaborative effort with his wife, Sabine, in Gerhard Wagner’s laboratory here in our department. The approach is to use fragment-based screening and NMR to identify small molecules that bind to specific regions of the primase.

CONNECTIONS WITH INDUSTRY My connections with industry have been rather limited compared with those of many of my colleagues. However, those few were enjoyable and educational. I agree with Arthur Kornberg, who remarked that it is tempting to take on consultations because they are much easier than research.

Hoffman–Roche Shortly after arriving at Harvard Medical School, Alex Nussbaum at Hoffman–Roche in Nutley, New Jersey, asked me to consult with his group. During my postdoctoral period Alex had spent a year at Stanford, where he developed skill in synthesizing oligonucleotides (269). Alex was a true Renaissance man. Before Google, the phrase was “Just ask Alex,” and one could do so in any of several languages, including Latin and Greek. After World War II he was the first graduate student of Carl Djerassi and went with him to Mexico to purify steroids from cactus, compounds that eventually led to the birth control pill and the founding of Syntex. Alex’s foresight on the impact of molecular biology was far ahead of most at Hoffman–Roche. He chemically synthesized DNA encoding the terminal redundancy of phage λDNA (270) as well as the S peptide of ribonuclease A (271). I consulted with Hoffman–Roche for several years, but I could do little to convince the 18

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senior group that there might be a future in recombinant DNA. Alex should have been in academia, and he did return to our department at Harvard for a sabbatical with George Fareed. Unwilling to return to Big Pharma, he joined the Boston Biomedical Foundation. He eventually became my laboratory director when I was chairman of the department.


United States Biochemical Corporation In November 1983, Thomas Mann, president of United States Biochemical Corporation (USB) in Cleveland, contacted me. Tom was attempting to establish a scientific board, and Paul Berg had suggested me as a potential member. Most likely I was one of the few remaining scientists with no consulting arrangements and therefore might be interested in a mundane company supplying biological products. I learned that USB produced few products of their own and did not supply enzymes used in recombinant DNA technology. However, Tom came with a consulting check in hand, and it was difficult to refuse. Tom Mann’s father founded Nutritional Biochemical Corporation just after World War II and received input from Jesse Greenstein and Milton Winitz at the NIH. The company was sold in 1969 to International Chemical Nuclear Corporation (ICN). In 1973 Tom founded USB, which developed a market supplying compounds for manufacturing diagnostic reagents and drugs, as well as for research laboratories. A few months after my association with USB, we recruited Bob Lehman as a member of the committee and, somewhat later, Fritz Eckstein, Stan Tabor, and Tom Cech. In short order, the company had the basic nucleic enzymes in stock, all prepared by Park Flick, a former student of Konrad Bloch and a postdoc with Roy Vagelos. Scientists are hesitant to change the source of their reagents. Consequently, sales did not dramatically increase. In 1985 Stan Tabor had described the T7 RNA polymerase system for gene expression, a system developed simultaneously by Bill Studier. The company introduced T7 RNA polymerase, bringing them to the attention of the scientific community. Shortly thereafter, beautiful sequencing gels were produced using chemically modified T7 DNA polymerase. We were fortunate that Richard Warburg, a member of the biochemistry department at Brandeis, was working part time at Fish & Richardson, a patent law firm in Boston. Harvard licensed the use of chemically modified T7 DNA polymerase to USB in 1986. Alex Nussbaum suggested the name Sequenase, and it soon became the enzyme of choice for DNA sequencing. Subsequently, Sequenase Version II, a genetically derived exonuclease-free enzyme, was introduced, and eventually Thermosequence was created to fulfill the needs of the automated sequencing machines. USB was sold to Amersham Life Sciences around this time. I remained as a consultant for several years, but the ability to make meaningful contributions decreased rapidly, as so often happens with quickly growing enterprises. However, I enjoyed many trips to England, where I stayed at the Hartwell House, the home of the ancestors of Henry “Light-Horse Harry” Lee and of Robert E. Lee. My association with USB was an enlightening and pleasurable chapter of my career. Tom Mann spared no expense to assure the success of the sequencing endeavor. Ingrid and I recall trips to France, Russia, and Japan, where the marketing of DNA-sequencing enzymes enabled us to travel on a scale we would never have considered. Tom Mann remains a close friend. At the time Sequenase was introduced, we were fortunate that Carl Fuller, a postdoctoral fellow in the lab, agreed to spend a few weeks in Cleveland to help with the enzyme preparation. He never left. Jack Chase, who discovered E. coli exonuclease VII in the early years of the lab and was then a professor at Albert Einstein College of Medicine, became scientific director. Denise Richardson, www.annualreviews.org • It Seems Like Only Yesterday

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a former technician in my lab, had moved to Cleveland, and she applied for a position at USB. Somewhat later, Bob Lechner joined the company to work on ribozymes in what became the genesis of Ribozyme Pharmaceuticals, the company founded by Tom Cech.

Genetics Institute, Inc.


In 1983 Mark Ptashne and Tom Maniatis asked me to consult with the protein development group at Genetics Institute, a biotechnology company they had founded. The group was relatively small, and the area of consultation was certainly within my expertise. Equally important was that the company was just across the street in the former Boston Lying-In Hospital, where my two sons were born. I continued this consultation for 20 years, eventually joining the company’s scientific board. It was educational to see the company evolve into a pharmaceutical company taking its protein therapeutics to clinical trial. The company moved to its new buildings in Cambridge, necessitating a longer commute, and the Process Development Group eventually moved to Andover, Massachusetts, forcing me to acquire a new car. American Home Products purchased Genetics Institute and, in turn, was acquired by Pfizer. This consultation even led to an entry into the rapidly expanding field of HIV-1 metabolism. Hans Huber in my lab collaborated with John McCoy and Jasbir Seehra at Genetics Institute to obtain some of the first definitive information on the kinetics of HIV-1 reverse transcriptase (272, 273). Some years later, Bruno Canard joined the lab and examined the binding of the enzyme to its RNA template and to nucleoside triphosphates (162–164).

SERENDIPITY Along the way there have been tangential but tantalizing projects. They present intriguing puzzles with no obvious solution, and they invariably lead to novel findings. On a recent grant application, one of the major criticisms was that I seem to be working on a particular project “out of curiosity.” Precisely! In this section I briefly discuss three of these projects.

Rediscovery of E. coli dGTPase The genes of T7 are clustered according to function. Some genes in the replication cluster are not essential, so the question arose as to duplication of function by a host protein. In 1981, Haruo Saito isolated a mutant of E. coli, optA1, that could not support a T7 phage lacking gene 1.2, located in the replication cluster (274). E. coli optA1 had no apparent defect in its growth. A genetic analysis revealed that the processing of gene 1.2 mRNA by ribonuclease III of E. coli regulates its expression—an interesting mechanism whereby the sequence in the hairpin that RNase III cleaves is complementary to the ribosome-binding site (275). Gene 1.1 lies upstream from gene 1.2, and the two proteins are transcribed from a single transcript. Kathy Ryan examined a chemically synthesized gene 1.1 protein and found it bound weakly to DNA, but no role has been assigned to it. Six years later, Jill Myers and Ben Beauchamp found that dGTP levels in optA1 mutants were extremely low (252, 253), leading to the purification of the product of the optA gene, a dGTPase (276). The enzyme is interesting both for its unusual physical properties and for the reaction it catalyzes. It is a dGTP triphosphohydrolase that cleaves dGTP to dGMP and tripolyphosphate. Susannah Wurgler found that the optA1 mutation resides in the promoter and increases expression (277). The protein binds extremely tightly to ssDNA (278). Sylvie Kornberg had found this enzyme in preparations of E. coli DNA polymerase in 1958 (279), and we had rediscovered it. Hans Huber 20

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showed that the gene 1.2 protein bound to and inhibited the dGTPase (280), and Hiroshi Nakai demonstrated that the complex of the two proteins forms a GTP-binding protein (281). Only in 2008, 50 years after its discovery, was a phenotype identified. Gawel, Hamilton, and Schaaper found that dGTPase-deficient mutants are novel mutators and that alterations of the dGTP pool may result in the formation of dGTP analogs (282).


A Novel Nucleotide Kinase An attempt by Stan Tabor to identify altered DNA polymerases that discriminate against dideoxynucleoside 5 -triphosphates (ddNTPs) led to a fascinating story. Recall that T7 DNA polymerase, unlike other members of this family, incorporates ddNTPs as well as dNTPs (deoxynucleoside triphosphates) (132, 145). The selection was for T7 phage that could grow in media containing dideoxynucleosides on the basis of the predicted phenotype of a T7 DNA polymerase that could not incorporate dideoxythymidine 5 -monophosphate (ddTMP). T7 phage resistant to inhibition by dideoxythymidine were isolated, but all the mutations resided in gene 1.7, a nonessential gene (256). After several years of frustration for others, Ngoc Tran found that the difficulty in purification was the precipitation of gene 1.7 protein at low ionic strength, such as 100 mM NaCl (234). The purified protein catalyzes the phosphorylation of dTMP and dGMP to the corresponding dNDP by using either dTTP or dGTP as the phosphate donor with an equilibrium near one (214). This unique nucleotide kinase readily phosphorylates ddTMP, whereas the host thymidylate kinase does not, thus explaining the sensitivity of T7 to dideoxythymidine. Unlike all other known nucleotide kinases, a divalent metal is not required and no nucleotidebinding motifs can be identified. The finding that this kinase is a dodecamer has increased our interest in a determination of its structure.

T7 Gene 5.5 Protein, E. coli H-NS, and Transfer RNA Priming The third fascinating story is that of the T7 gene 5.5 protein. The proximity of gene 5.5 to gene 5, which encodes DNA polymerase, suggests the possibility that the proteins interact. In 1993, Qingyun Liu (241) found that gene 5.5 protein purified as a tight complex with E. coli H-NS protein. H-NS binds to double-stranded DNA, particularly bent or curved DNA; condenses the DNA; and results in global gene regulation (283). Almost 20 years, later Bin Zhu and colleagues in my lab (240) identified mutations in gene 5.5 that allowed T7 phage, which lacked the ability to synthesize RNA primers, to nevertheless grow in E. coli. Subsequent studies revealed that gene 5.5 protein binds to E. coli transfer RNA (tRNA) and that this complex sequesters the H-NS protein. In the absence of gene 5.5 protein, there is an abundance of tRNA, and the 5 -ACCA-3 C terminus of the tRNA corresponds to one of the primers synthesized by the T7 primase. The tRNA binds to a primase recognition sequence under the direction of the defective DNA primase and primes the synthesis of Okazaki fragments. Thus, tRNA priming occurs in prokaryotic systems, as well as in some eukaryotic viruses such as HIV-1 (284).

ANNUAL REVIEW OF BIOCHEMISTRY: A PLEASANT TASK In 1969 I wrote a review for the Annual Review of Biochemistry titled “Enzymes in DNA Metabolism” (285). You could do that then but not today. I found the time away from the bench irksome and swore never to accept such an invitation again. However, I did, much later, contribute two additional reviews to the Annual Review of Biochemistry for which postdoctoral fellows David Frick www.annualreviews.org • It Seems Like Only Yesterday

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(148) and Sam Hamdan (149), respectively, did much of the scut work. Now, here I am again contributing a fourth but, like the first, without any assistance. I joined the Annual Review of Biochemistry in 1972 as an Associate Editor upon the resignation of Bob Sinsheimer. Esmond Snell was the Editor, and the Associate Editors were Paul Boyer, Alton Meister, and myself. I became Editor upon the resignation of Esmond Snell in 1983 and remained in that position until I resigned in 2003. The positions of Associate Editor and Editor were not only a privilege but also educational, enjoyable, and a way to spend a day with the leaders of all fields of biochemistry. Essentially all of the work was accomplished on that single day when we held the annual meeting, often at very pleasant locations: Palo Alto, San Francisco, Napa Valley, La Jolla, New Mexico, New Orleans, Boston, Duke University, Seattle, Hawaii. The Annual Review of Biochemistry had a very high rate of acceptance of invitations. I considered it an honor when my invitations came, unsolicited, from my colleagues. Although my colleagues would, on occasion, volunteer to contribute a review, all were treated the same. At the annual meeting, the Editorial Committee made suggestions for topics and potential authors; the names of volunteers were added to the list if the committee so decided. Roger Kornberg assumed the editorship upon my departure, and it could not have passed into better hands.


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EPILOGUE In October 2005 I was honored with a symposium upon my seventieth birthday. Nearly all the current and previous members of my laboratory, as well as colleagues from over the years and their spouses, met for three days at the Chatham Bars Inn on Cape Cod. It was a wonderful but somewhat overwhelming occasion to see everyone again and to realize they had all more than fulfilled my expectations for their careers. One such celebration is sufficient for me, so I take this opportunity to express to all the students and research fellows, both past and present, as well as my colleagues in academia and business, my sincere appreciation for all the contributions they have made to my research program. Even more important have been the personal interactions with them and their families. These friendships have significantly enriched my life. Finally, my entire career has depended on the generous funding provided by the National Institutes of Health, the American Cancer Society, and the Department of Energy. I intend to phase out the lab in 2016, and I have yet to decide precisely what I will do henceforth. I am tempted to keep a bench, and in relative solitude I might have the courage to once again undertake some hands-on experiments on long-neglected but favorite projects.

DISCLOSURE STATEMENT The author is not aware of any affiliations, memberships, funding, or financial holdings that might be perceived as affecting the objectivity of this review. LITERATURE CITED 1. Jukes TH. 1983. Philip Handler (1917–1981). A biographical sketch. J. Nutr. 113:1086–94 2. White A, Handler P, Smith EL, Stetten D, ed. 1954. Principles of Biochemistry. New York: McGraw-Hill 3. Avery OT, Macleod CM, McCarty M. 1944. Studies on the chemical nature of the substance inducing transformation of pneumococcal types: induction of transformation by a deoxyribonucleic acid fraction isolated from Pneumococcus type III. J. Exp. Med. 79:137–58 4. Lehman IR, Bessman MJ, Simms ES, Kornberg A. 1958. Enzymatic synthesis of deoxyribonucleic acid. I. Preparation of substrates and partial purification of an enzyme from Escherichia coli. J. Biol. Chem. 233:163–70 22

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5. Schwert GW. 1958. The mode of action of heart muscle lactic dehydrogenase. Ann. N.Y. Acad. Sci. 75:311–13 6. Wyngaarden JB, Ashton DM. 1959. Feedback control of purine biosynthesis by purine ribonucleotides. Nature 183:747–48 7. Laszlo J, Neelon FA. 2006. The Doctor’s Doctor: A Biography of Eugene A. Stead Jr., MD. Durham, NC: Carolina Acad. 346 pp. 8. Stead EA, Wagner GS, Cebe B, Rozear MP. 1978. E.A. Stead, Jr.: What This Patient Needs Is a Doctor. Durham, NC: Carolina Acad. 244 pp. 9. Kornberg A. 1989. Never a dull enzyme. Annu. Rev. Biochem. 58:1–30 10. Richardson CC, Schildkraut CL, Aposhian HV, Kornberg A. 1964. Enzymatic synthesis of deoxyribonucleic acid. XIV. Further purification and properties of deoxyribonucleic acid polymerase of Escherichia coli. J. Biol. Chem. 239:222–32 11. Gladwell M. 2008. Outliers: The Story of Success. New York: Little, Brown 12. Richardson CC, Schildkraut CL, Aposhian HV, Kornberg A, Bodmer W, Lederberg J. 1963. Studies on the Replication of DNA by Escherichia coli Polymerase. New York: Academic 13. Richardson CC, Kornberg A. 1964. A deoxyribonucleic acid phosphatase-exonuclease from Escherichia coli. I. Purification of the enzyme and characterization of the phosphatase activity. J. Biol. Chem. 239:242– 50 14. Richardson CC, Lehman IR, Kornberg A. 1964. A deoxyribonucleic acid phosphatase-exonuclease from Escherichia coli. II. Characterization of the exonuclease activity. J. Biol. Chem. 239:251–58 15. Richardson CC, Inman RB, Kornberg A. 1964. Enzymic synthesis of deoxyribonucleic acid. 18. The repair of partially single-stranded DNA templates by DNA polymerase. J. Mol. Biol. 9:46–69 16. Schildkraut CL, Richardson CC, Kornberg A. 1964. Enzymic synthesis of deoxyribonucleic acid. XVII. Some unusual physical properties of the product primed by native DNA templates. J. Mol. Biol. 9:24–45 17. Lehman IR, Richardson CC. 1964. The deoxyribonucleases of Escherichia coli. IV. An exonuclease activity present in purified preparations of deoxyribonucleic acid polymerase. J. Biol. Chem. 239:233–41 18. Luria SE. 1953. Host-induced modifications of viruses. Cold Spring Harb. Symp. Quant. Biol. 18:237–44 19. Richardson CC. 1966. Influence of glucosylation of deoxyribonucleic acid on hydrolysis by deoxyribonucleases of Escherichia coli. J. Biol. Chem. 241:2084–92 20. Lehman IR. 1960. The deoxyribonucleases of Escherichia coli. I. Purification and properties of a phosphodiesterase. J. Biol. Chem. 235:1479–87 21. MacHattie LA, Ritchie DA, Thomas CA Jr, Richardson CC. 1967. Terminal repetition in permuted T2 bacteriophage DNA molecules. J. Mol. Biol. 23:355–63 22. Fleischman RA, Richardson CC. 1971. Analysis of host range restriction in Escherichia coli treated with toluene. PNAS 68:2527–31 23. Fleischman RA, Campbell JL, Richardson CC. 1976. Modification and restriction of T-even bacteriophages. In vitro degradation of deoxyribonucleic acid containing 5-hydroxymethylctosine. J. Biol. Chem. 251:1561–70 24. Okazaki R, Okazaki T, Sakabe K, Sugimoto K, Sugino A. 1968. Mechanism of DNA chain growth. I. Possible discontinuity and unusual secondary structure of newly synthesized chains. PNAS 59:598–605 25. Richardson CC. 1965. Phosphorylation of nucleic acid by an enzyme from T4 bacteriophage–infected Escherichia coli. PNAS 54:158–65 26. Novogrodsky A, Hurwitz J. 1966. The enzymatic phosphorylation of ribonucleic acid and deoxyribonucleic acid. I. Phosphorylation at 5 -hydroxyl termini. J. Biol. Chem. 241:2923–32 27. Richardson CC. 1966. The 5 -terminal nucleotides of T7 bacteriophage deoxyribonucleic acid. J. Mol. Biol. 15:49–61 28. Dunn JJ, Studier FW. 1983. Complete nucleotide sequence of bacteriophage T7 DNA and the locations of T7 genetic elements. J. Mol. Biol. 166:477–535 29. Weiss B, Richardson CC. 1967. The 5 -terminal dinucleotides of the separated strands of T7 bacteriophage deoxyribonucleic acid. J. Mol. Biol. 23:405–15 30. Weiss B, Live TR, Richardson CC. 1968. Enzymatic breakage and joining of deoxyribonucleic acid. V. End group labeling and analysis of deoxyribonucleic acid containing single stranded breaks. J. Biol. Chem. 243:4530–42 www.annualreviews.org • It Seems Like Only Yesterday

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31. Weiss B, Richardson CC. 1966. End-group labeling of nucleic acids by enzymatic phosphorylation. Cold Spring Harb. Symp. Quant. Biol. 31:471–78 32. Richardson CC, Weiss B. 1966. The enzymatic phosphorylation of nucleic acids and its application to end-group analysis. J. Gen. Physiol. 49:81–97 33. Masamune Y, Fleischman RA, Richardson CC. 1971. Enzymatic removal and replacement of nucleotides at single strand breaks in deoxyribonucleic acid. J. Biol. Chem. 246:2680–91 34. Masamune Y, Richardson CC. 1971. Strand displacement during deoxyribonucleic acid synthesis at single strand breaks. J. Biol. Chem. 246:2692–701 35. Scheffler IE, Richardson CC. 1972. Chemical and enzymatic studies of deoxyribonucleic acid covalently linked to Ficoll. J. Biol. Chem. 247:5736–45 36. Weiss B, Richardson CC. 1967. Enzymatic breakage and joining of deoxyribonucleic acid. I. Repair of single-strand breaks in DNA by an enzyme system from Escherichia coli infected with T4 bacteriophage. PNAS 57:1021–28 37. Richardson CC, Masamune Y, Live TR, Jacquemin-Sablon A, Weiss B, Fareed GC. 1968. Studies on the joining of DNA by polynucleotide ligase of phage T4. Cold Spring Harb. Symp. Quant. Biol. 33:151–64 38. Weiss B, Jacquemin-Sablon A, Live TR, Fareed GC, Richardson CC. 1968. Enzymatic breakage and joining of deoxyribonucleic acid. VI. Further purification and properties of polynucleotide ligase from Escherichia coli infected with bacteriophage T4. J. Biol. Chem. 243:4543–55 39. Weiss B, Thompson A, Richardson CC. 1968. Ezymatic breakage and joining of deoxyribonucleic acid. VII. Properties of the enzyme-adenylate intermediate in the polynucleotide ligase reaction. J. Biol. Chem. 243:4556–63 40. Harvey CL, Gabriel TF, Wilt EM, Richardson CC. 1971. Enzymatic breakage and joining of deoxyribonucleic acid. IX. Synthesis and properties of the deoxyribonucleic acid adenylate in the phage T4 ligase reaction. J. Biol. Chem. 246:4523–30 41. Fareed GC, Wilt EM, Richardson CC. 1971. Enzymatic breakage and joining of deoxyribonucleic acid. 8. Hybrids of ribo- and deoxyribonucleotide homopolymers as substrates for polynucleotide ligase of bacteriophage T4. J. Biol. Chem. 246:925–32 42. Zimmerman SB, Little JW, Oshinsky CK, Gellert M. 1967. Enzymatic joining of DNA strands: a novel reaction of diphosphopyridine nucleotide. PNAS 57:1841–48 43. Olivera BM, Lehman IR. 1967. Linkage of polynucleotides through phosphodiester bonds by an enzyme from Escherichia coli. PNAS 57:1426–33 44. Gefter ML, Becker A, Hurwitz J. 1967. The enzymatic repair of DNA. I. Formation of circular λ-DNA. PNAS 58:240–47 45. Cozzarelli NR, Melechen NE, Jovin TM, Kornberg A. 1967. Polynucleotide cellulose as a substrate for a polynucleotide ligase induced by phage T4. Biochem. Biophys. Res. Commun. 28:578–86 46. Fareed GC, Richardson CC. 1967. Enzymatic breakage and joining of deoxyribonucleic acid. II. The structural gene for polynucleotide ligase in bacteriophage T4. PNAS 58:665–72 47. Masamune Y, Frenkel GD, Richardson CC. 1971. A mutant of bacteriophage T7 deficient in polynucleotide ligase. J. Biol. Chem. 246:6874–79 48. Jacquemin-Sablon A, Richardson CC. 1970. Analysis of the interruptions in bacteriophage T5 DNA. J. Mol. Biol. 47:477–93 49. Frenkel GD, Richardson CC. 1971. The deoxyribonuclease induced after infection of Escherichia coli by bacteriophage T5. II. Role of the enzyme in replication of the pahge deoxyribonucleic acid. J. Biol. Chem. 246:4848–52 50. Frenkel GD, Richardson CC. 1971. The deoxyribonuclease induced after infection of Escherichia coli by bacteriophage T5. I. Characterization of the enzyme as a 5 -exonuclease. J. Biol. Chem. 246:4839–47 51. Paul AV, Lehman IR. 1966. The deoxyribonucleases of Escherichia coli. VII. A deoxyribonuclease induced by infection with phage T-5. J. Biol. Chem. 241:3441–51 52. Khorana HG. 1979. Total synthesis of a gene. Science 203:614–25 53. Hirota Y, Mordoh J, Scheffler I, Jacob F. 1972. Genetic approach to DNA replication and its control in Escherichia coli. Fed. Proc. 31:1422–27 54. De Lucia P, Cairns J. 1969. Isolation of an E. coli strain with a mutation affecting DNA polymerase. Nature 224:1164–66


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55. Moses RE, Richardson CC. 1970. Replication and repair of DNA in cells of Escherichia coli treated with toluene. PNAS 67:674–81 56. Moses RE, Campbell JL, Fleischman RA, Frenkel GD, Mulcahy HL, et al. 1972. Enzymatic mechanisms of DNA replication in Escherichia coli. Fed. Proc. 31:1415–21 57. Moses RE, Richardson CC. 1970. A new DNA polymerase acitvity of Escherichia coli. II. Properties of the enzyme purified from wild-type E. coli and DNA-ts mutants. Biochem. Biophys. Res. Commun. 41:1565–71 58. Moses RE, Richardson CC. 1970. A new DNA polymerase activity of Escherichia coli. I. Purification and properties of the activity present in E. coli polA1. Biochem. Biophys. Res. Commun. 41:1557–64 59. Campbell JL, Shizuya H, Richardson CC. 1974. Mapping of a mutation, polB100, affecting deoxyribonucleic acid polymerase II in Escherichia coli K-12. J. Bacteriol. 119:494–99 60. Campbell JL, Soll L, Richardson CC. 1972. Isolation and partial characterization of a mutant of Escherichia coli deficient in DNA polymerase II. PNAS 69:2090–94 61. Hirota Y, Gefter ML, Mindich L. 1972. A mutant of Escherichia coli defective in DNA polymerase II activity. PNAS 69:3238–42 62. Kornberg T, Gefter ML. 1970. DNA synthesis in cell-free extracts of a DNA polymerase–defective mutant. Biochem. Biophys. Res. Commun. 40:1348–55 63. Kornberg T, Gefter ML. 1971. Purification and DNA synthesis in cell-free extracts: properties of DNA polymerase II. PNAS 68:761–64 64. Gefter ML, Hirota Y, Kornberg T, Wechsler JA, Barnoux C. 1971. Analysis of DNA polymerases II and III in mutants of Escherichia coli thermosensitive for DNA synthesis. PNAS 68:3150–53 65. Moore FD. 1973. Edward Delos Churchill: 1895–1972. Ann. Surg. 177:507–8 66. Livingston DM, Hinkle DC, Richardson CC. 1975. Deoxyribonucleic acid polymerase III of Escherichia coli. Purification and properties. J. Biol. Chem. 250:461–69 67. Livingston DM, Richardson CC. 1975. Deoxyribonucleic acid polymerase III of Escherichia coli. Characterization of associated exonuclease activities. J. Biol. Chem. 250:470–78 68. Chase JW, Richardson CC. 1977. Escherichia coli mutants deficient in exonuclease VII. J. Bacteriol. 129:934–47 69. Chase JW, Richardson CC. 1975. Exonuclease VII of Escherichia coli. Basic Life Sci. 5A:225–34 70. Chase JW, Richardson CC. 1974. Exonuclease VII of Escherichia coli. Mechanism of action. J. Biol. Chem. 249:4553–61 71. Chase JW, Richardson CC. 1974. Exonuclease VII of Escherichia coli. Purification and properties. J. Biol. Chem. 249:4545–52 72. Shizuya H, Richardson CC. 1974. Synthesis of bacteriophage λDNA in vitro: requirement for O and P gene products. PNAS 71:1758–62 73. Studier FW. 1972. Bacteriophage T7. Science 176:367–76 74. Studier FW. 1969. The genetics and physiology of bacteriophage T7. Virology 39:562–74 75. Center MS, Richardson CC. 1970. An endonuclease induced after infection of Escherichia coli with bacteriophage T7. II. Specificity of the enzyme toward single- and double-stranded deoxyribonucleic acid. J. Biol. Chem. 245:6292–99 76. Center MS, Richardson CC. 1970. An endonuclease induced after infection of Escherichia coli with bacteriophage T7. I. Purification and properties of the enzyme. J. Biol. Chem. 245:6285–91 77. Center MS, Studier FW, Richardson CC. 1970. The structural gene for a T7 endonuclease essential for phage DNA synthesis. PNAS 65:242–48 78. Grippo P, Richardson CC. 1971. Deoxyribonucleic acid polymerase of bacteriophage T7. J. Biol. Chem. 246:6867–73 79. Masamune Y, Richardson CC. 1968. Enzymatic breakage and joining of deoxyribonucleic acid. IV. DNA synthesis in E. coli infected with ligase-negative mutants of phage T4. PNAS 61:1328–35 80. LeClerc JE, Richardson CC. 1979. Gene 2 protein of bacteriophage T7: purification and requirement for packaging of T7 DNA in vitro. PNAS 76:4852–56 81. Campbell JL, Tamanoi F, Richardson CC, Studier FW. 1979. Cloning of the T7 genome in Escherichia coli: use of recombination between cloned sequences and bacteriophage T7 to identify genes involved in recombination and a clone containing the origin of T7 DNA replication. Cold Spring Harb. Symp. Quant. Biol. 43:441–48 www.annualreviews.org • It Seems Like Only Yesterday

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175. Park K, Debyser Z, Tabor S, Richardson CC, Griffith JD. 1998. Formation of a DNA loop at the replication fork generated by bacteriophage T7 replication proteins. J. Biol. Chem. 273:5260–70 176. Frick DN, Kumar S, Richardson CC. 1999. Interaction of ribonucleoside triphosphates with the gene 4 primase of bacteriophage T7. J. Biol. Chem. 274:35899–907 177. Frick DN, Richardson CC. 1999. Interaction of bacteriophage T7 gene 4 primase with its template recognition site. J. Biol. Chem. 274:35889–98 178. Kato M, Frick DN, Lee J, Tabor S, Richardson CC, Ellenberger T. 2001. A complex of the bacteriophage T7 primase–helicase and DNA polymerase directs primer utilization. J. Biol. Chem. 276:21809–20 179. Tseng TY, Frick DN, Richardson CC. 2000. Characterization of a novel DNA primase from the Salmonella typhimurium bacteriophage SP6. Biochemistry 39:1643–54 180. Ghosh S, Hamdan SM, Cook TE, Richardson CC. 2008. Interactions of Escherichia coli thioredoxin, the processivity factor, with bacteriophage T7 DNA polymerase and helicase. J. Biol. Chem. 283:32077–84 181. Ghosh S, Hamdan SM, Richardson CC. 2010. Two modes of interaction of the single-stranded DNA– binding protein of bacteriophage T7 with the DNA polymerase–thioredoxin complex. J. Biol. Chem. 285:18103–12 182. Ghosh S, Marintcheva B, Takahashi M, Richardson CC. 2009. C-terminal phenylalanine of bacteriophage T7 single-stranded DNA–binding protein is essential for strand displacement synthesis by T7 DNA polymerase at a nick in DNA. J. Biol. Chem. 284:30339–49 183. Satapathy AK, Kulczyk AW, Ghosh S, van Oijen AM, Richardson CC. 2011. Coupling dTTP hydrolysis with DNA unwinding by the DNA helicase of bacteriophage T7. J. Biol. Chem. 286:34468–78 184. Guo S, Tabor S, Richardson CC. 1999. The linker region between the helicase and primase domains of the bacteriophage T7 gene 4 protein is critical for hexamer formation. J. Biol. Chem. 274:30303–9 185. Sawaya MR, Guo S, Tabor S, Richardson CC, Ellenberger T. 1999. Crystal structure of the helicase domain from the replicative helicase–primase of bacteriophage T7. Cell 99:167–77 186. Etson CM, Hamdan SM, Richardson CC, van Oijen AM. 2010. Thioredoxin suppresses microscopic hopping of T7 DNA polymerase on duplex DNA. PNAS 107:1900–5 187. Hamdan SM, Johnson DE, Tanner NA, Lee JB, Qimron U, et al. 2007. Dynamic DNA helicase–DNA polymerase interactions assure processive replication fork movement. Mol. Cell 27:539–49 188. Hamdan SM, Loparo JJ, Takahashi M, Richardson CC, van Oijen AM. 2009. Dynamics of DNA replication loops reveal temporal control of lagging-strand synthesis. Nature 457:336–39 189. Hamdan SM, Marintcheva B, Cook T, Lee SJ, Tabor S, Richardson CC. 2005. A unique loop in T7 DNA polymerase mediates the binding of helicase–primase, DNA binding protein, and processivity factor. PNAS 102:5096–101 190. Johnson DE, Takahashi M, Hamdan SM, Lee SJ, Richardson CC. 2007. Exchange of DNA polymerases at the replication fork of bacteriophage T7. PNAS 104:5312–17 191. Lee JB, Hite RK, Hamdan SM, Xie XS, Richardson CC, van Oijen AM. 2006. DNA primase acts as a molecular brake in DNA replication. Nature 439:621–24 192. Lee SJ, Marintcheva B, Hamdan SM, Richardson CC. 2006. The C-terminal residues of bacteriophage T7 gene 4 helicase–primase coordinate helicase and DNA polymerase activities. J. Biol. Chem. 281:25841– 49 193. Lee SJ, Zhu B, Hamdan SM, Richardson CC. 2010. Mechanism of sequence-specific template binding by the DNA primase of bacteriophage T7. Nucleic Acids Res. 38:4372–83 194. Marintcheva B, Hamdan SM, Lee SJ, Richardson CC. 2006. Essential residues in the C terminus of the bacteriophage T7 gene 2.5 single-stranded DNA–binding protein. J. Biol. Chem. 281:25831–40 195. Qimron U, Kulczyk AW, Hamdan SM, Tabor S, Richardson CC. 2008. Inadequate inhibition of host RNA polymerase restricts T7 bacteriophage growth on hosts overexpressing UDK. Mol. Microbiol. 67:448–57 196. Qimron U, Lee SJ, Hamdan SM, Richardson CC. 2006. Primer initiation and extension by T7 DNA primase. EMBO J. 25:2199–208 197. Scholle MD, Banach BS, Hamdan SM, Richardson CC, Kay BK. 2008. Peptide ligands specific to the oxidized form of Escherichia coli thioredoxin. Biochim. Biophys. Acta 1784:1735–41


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222. Kong D, Nossal NG, Richardson CC. 1997. Role of the bacteriophage T7 and T4 single-stranded DNA–binding proteins in the formation of joint molecules and DNA helicase–catalyzed polar branch migration. J. Biol. Chem. 272:8380–87 223. Kong D, Richardson CC. 1998. Role of the acidic carboxyl-terminal domain of the single-stranded DNA– binding protein of bacteriophage T7 in specific protein–protein interactions. J. Biol. Chem. 273:6556–64 224. Kong D, Richardson CC. 1996. Single-stranded DNA binding protein and DNA helicase of bacteriophage T7 mediate homologous DNA strand exchange. EMBO J. 15:2010–19 225. Kumar JK, Tabor S, Richardson CC. 2001. Role of the C-terminal residue of the DNA polymerase of bacteriophage T7. J. Biol. Chem. 276:34905–12 226. Lee J, Chastain PD 2nd, Griffith JD, Richardson CC. 2002. Lagging strand synthesis in coordinated DNA synthesis by bacteriophage T7 replication proteins. J. Mol. Biol. 316:19–34 227. Notarnicola SM, Mulcahy HL, Lee J, Richardson CC. 1997. The acidic carboxyl terminus of the bacteriophage T7 gene 4 helicase/primase interacts with T7 DNA polymerase. J. Biol. Chem. 272:18425–33 228. Lee SJ, Qimron U, Richardson CC. 2008. Communication between subunits critical to DNA binding by hexameric helicase of bacteriophage T7. PNAS 105:8908–13 229. Lee SJ, Richardson CC. 2010. Molecular basis for recognition of nucleoside triphosphate by gene 4 helicase of bacteriophage T7. J. Biol. Chem. 285:31462–71 230. Lee SJ, Richardson CC. 2005. Acidic residues in the nucleotide-binding site of the bacteriophage T7 DNA primase. J. Biol. Chem. 280:26984–91 231. Lee SJ, Richardson CC. 2004. The linker region between the helicase and primase domains of the gene 4 protein of bacteriophage T7. Role in helicase conformation and activity. J. Biol. Chem. 279:23384–93 232. Lee SJ, Richardson CC. 2002. Interaction of adjacent primase domains within the hexameric gene 4 helicase–primase of bacteriophage T7. PNAS 99:12703–8 233. Lee SJ, Richardson CC. 2001. Essential lysine residues in the RNA polymerase domain of the gene 4 primase–helicase of bacteriophage T7. J. Biol. Chem. 276:49419–26 234. Tran NQ, Lee SJ, Richardson CC, Tabor S. 2010. A novel nucleotide kinase encoded by gene 1.7 of bacteriophage T7. Mol. Microbiol. 77:492–504 235. Zhang H, Lee SJ, Richardson CC. 2012. The roles of tryptophans in primer synthesis by the DNA primase of bacteriophage T7. J. Biol. Chem. 287:23644–56 236. Zhang H, Lee SJ, Zhu B, Tran NQ, Tabor S, Richardson CC. 2011. Helicase–DNA polymerase interaction is critical to initiate leading-strand DNA synthesis. PNAS 108:9372–77 237. Zhu B, Lee SJ, Richardson CC. 2011. Bypass of a nick by the replisome of bacteriophage T7. J. Biol. Chem. 286:28488–97 238. Zhu B, Lee SJ, Richardson CC. 2010. Direct role for the RNA polymerase domain of T7 primase in primer delivery. PNAS 107:9099–104 239. Zhu B, Lee SJ, Richardson CC. 2009. An in trans interaction at the interface of the helicase and primase domains of the hexameric gene 4 protein of bacteriophage T7 modulates their activities. J. Biol. Chem. 284:23842–51 240. Zhu B, Lee SJ, Tan M, Wang ED, Richardson CC. 2012. Gene 5.5 protein of bacteriophage T7 in complex with Escherichia coli nucleoid protein H-NS and transfer RNA masks transfer RNA priming in T7 DNA replication. PNAS 109:8050–55 241. Liu Q, Richardson CC. 1993. Gene 5.5 protein of bacteriophage T7 inhibits the nucleoid protein H-NS of Escherichia coli. PNAS 90:1761–65 242. Marintcheva B, Marintchev A, Wagner G, Richardson CC. 2008. Acidic C-terminal tail of the ssDNAbinding protein of bacteriophage T7 and ssDNA compete for the same binding surface. PNAS 105:1855– 60 243. Marintcheva B, Qimron U, Yu Y, Tabor S, Richardson CC. 2009. Mutations in the gene 5 DNA polymerase of bacteriophage T7 suppress the dominant lethal phenotype of gene 2.5 ssDNA binding protein lacking the C-terminal phenylalanine. Mol. Microbiol. 72:869–80 244. Qimron U, Marintcheva B, Tabor S, Richardson CC. 2006. Genomewide screens for Escherichia coli genes affecting growth of T7 bacteriophage. PNAS 103:19039–44


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245. Shokri L, Marintcheva B, Richardson CC, Rouzina I, Williams MC. 2006. Single molecule force spectroscopy of salt-dependent bacteriophage T7 gene 2.5 protein binding to single-stranded DNA. J. Biol. Chem. 281:38689–96 246. Mendelman LV, Beauchamp BB, Richardson CC. 1994. Requirement for a zinc motif for template recognition by the bacteriophage T7 primase. EMBO J. 13:3909–16 247. Mendelman LV, Kuimelis RG, McLaughlin LW, Richardson CC. 1995. Effects of base analog substitutions in the noncoding dC of the 3 -d(CTG)-5 template recognition site of the bacteriophage T7 primase. Biochemistry 34:10187–93 248. Mendelman LV, Notarnicola SM, Richardson CC. 1993. Evidence for distinct primase and helicase domains in the 63-kDa gene 4 protein of bacteriophage T7. Characterization of nucleotide binding site mutant. J. Biol. Chem. 268:27208–13 249. Mendelman LV, Notarnicola SM, Richardson CC. 1992. Roles of bacteriophage T7 gene 4 proteins in providing primase and helicase functions in vivo. PNAS 89:10638–42 250. Mendelman LV, Richardson CC. 1991. Requirements for primer synthesis by bacteriophage T7 63-kDa gene 4 protein. Roles of template sequence and T7 56-kDa gene 4 protein. J. Biol. Chem. 266:23240–50 251. Mitsunobu H, Zhu B, Lee SJ, Tabor S, Richardson CC. 2014. Flap endonuclease activity of gene 6 exonuclease of bacteriophage T7. J. Biol. Chem. 289:5860–75 252. Myers JA, Beauchamp BB, Richardson CC. 1987. Gene 1.2 protein of bacteriophage T7. Effect on deoxyribonucleotide pools. J. Biol. Chem. 262:5288–92 253. Myers JA, Beauchamp BB, White JH, Richardson CC. 1987. Purification and characterization of the gene 1.2 protein of bacteriophage T7. J. Biol. Chem. 262:5280–87 254. Notarnicola SM, Park K, Griffith JD, Richardson CC. 1995. A domain of the gene 4 helicase/primase of bacteriophage T7 required for the formation of an active hexamer. J. Biol. Chem. 270:20215–24 255. Notarnicola SM, Richardson CC. 1993. The nucleotide binding site of the helicase/primase of bacteriophage T7. Interaction of mutant and wild-type proteins. J. Biol. Chem. 268:27198–207 256. Tran NQ, Rezende LF, Qimron U, Richardson CC, Tabor S. 2008. Gene 1.7 of bacteriophage T7 confers sensitivity of phage growth to dideoxythymidine. PNAS 105:9373–78 257. Rezende LF, Hollis T, Ellenberger T, Richardson CC. 2002. Essential amino acid residues in the singlestranded DNA–binding protein of bacteriophage T7. Identification of the dimer interface. J. Biol. Chem. 277:50643–53 258. Rezende LF, Willcox S, Griffith JD, Richardson CC. 2003. A single-stranded DNA–binding protein of bacteriophage T7 defective in DNA annealing. J. Biol. Chem. 278:29098–105 259. Scholl D, Kieleczawa J, Kemp P, Rush J, Richardson CC, et al. 2004. Genomic analysis of bacteriophages SP6 and K1-5, an estranged subgroup of the T7 supergroup. J. Mol. Biol. 335:1151–71 260. Satapathy AK, Richardson CC. 2011. The glutamate switch of bacteriophage T7 DNA helicase: role in coupling nucleotide triphosphate (NTP) and DNA binding to NTP hydrolysis. J. Biol. Chem. 286:23113– 20 261. Hollis T, Stattel JM, Walther DS, Richardson CC, Ellenberger T. 2001. Structure of the gene 2.5 protein, a single-stranded DNA binding protein encoded by bacteriophage T7. PNAS 98:9557–62 262. Tran NQ, Tabor S, Richardson CC. 2014. Genetic requirements for sensitivity of bacteriophage T7 to dideoxythymidine. J. Bacteriol. 196:2842–50 263. Zhu B, Tabor S, Richardson CC. 2014. Syn5 RNA polymerase synthesizes precise run-off RNA products. Nucleic Acids Res. 42:e33 264. Ritchie DA, Thomas CA Jr, MacHattie LA, Wensink PC. 1967. Terminal repetition in non-permuted T3 and T7 bacteriophage DNA molecules. J. Mol. Biol. 23:365–76 265. Doublie S, Tabor S, Long AM, Richardson CC, Ellenberger T. 1998. Crystal structure of a bacteriophage T7 DNA replication complex at 2.2 A˚ resolution. Nature 391:251–58 266. Kato M, Ito T, Wagner G, Richardson CC, Ellenberger T. 2003. Modular architecture of the bacteriophage T7 primase couples RNA primer synthesis to DNA synthesis. Mol. Cell 11:1349–60 267. Toth EA, Li Y, Sawaya MR, Cheng Y, Ellenberger T. 2003. The crystal structure of the bifunctional primase–helicase of bacteriophage T7. Mol. Cell 12:1113–23 268. Alberts BM, Barry J, Bedinger P, Formosa T, Jongeneel CV, Kreuzer KN. 1983. Studies on DNA replication in the bacteriophage T4 in vitro system. Cold Spring Harb. Symp. Quant. Biol. 47:655–68 www.annualreviews.org • It Seems Like Only Yesterday

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269. Lehman IR, Nussbaum AL. 1964. The deoxyribonucleases of Escherichia coli. V. On the specificity of exonuclease I (phosphodiesterase). J. Biol. Chem. 239:2628–36 270. Harvey CL, Wright R, Nussbaum AL. 1973. Lambda phage DNA: joining of a chemically synthesized cohesive end. Science 179:291–93 271. Harvey CL, Olson K, de Czekala A, Nussbaum AL. 1975. Construction of a double-stranded deoxyribonucleotide sequence of 45 base pairs designed to code for S-peptide 2-14 of bovine ribonuclease A. Nucleic Acids Res. 2:2007–20 272. Huber HE, McCoy JM, Seehra JS, Richardson CC. 1989. Human immunodeficiency virus 1 reverse transcriptase. Template binding, processivity, strand displacement synthesis, and template switching. J. Biol. Chem. 264:4669–78 273. Huber HE, Richardson CC. 1990. Processing of the primer for plus strand DNA synthesis by human immunodeficiency virus 1 reverse transcriptase. J. Biol. Chem. 265:10565–73 274. Saito H, Richardson CC. 1981. Genetic analysis of gene 1.2 of bacteriophage T7: isolation of a mutant of Escherichia coli unable to support the growth of T7 gene 1.2 mutants. J. Virol. 37:343–51 275. Saito H, Richardson CC. 1981. Processing of mRNA by ribonuclease III regulates expression of gene 1.2 of bacteriophage T7. Cell 27:533–42 276. Beauchamp BB, Richardson CC. 1988. A unique deoxyguanosine triphosphatase is responsible for the optA1 phenotype of Escherichia coli. PNAS 85:2563–67 277. Wurgler SM, Richardson CC. 1990. Structure and regulation of the gene for dGTP triphosphohydrolase from Escherichia coli. PNAS 87:2740–44 278. Wurgler SM, Richardson CC. 1993. DNA binding properties of the deoxyguanosine triphosphate triphosphohydrolase of Escherichia coli. J. Biol. Chem. 268:20046–54 279. Kornberg SR, Lehman IR, Bessman MJ, Simms ES, Kornberg A. 1958. Enzymatic cleavage of deoxyguanosine triphosphate to deoxyguanosine and tripolyphosphate. J. Biol. Chem. 233:159–62 280. Huber HE, Beauchamp BB, Richardson CC. 1988. Escherichia coli dGTP triphosphohydrolase is inhibited by gene 1.2 protein of bacteriophage T7. J. Biol. Chem. 263:13549–56 281. Nakai H, Richardson CC. 1990. The gene 1.2 protein of bacteriophage T7 interacts with the Escherichia coli dGTP triphosphohydrolase to form a GTP-binding protein. J. Biol. Chem. 265:4411–19 282. Gawel D, Hamilton MD, Schaaper RM. 2008. A novel mutator of Escherichia coli carrying a defect in the dgt gene, encoding a dGTP triphosphohydrolase. J. Bacteriol. 190:6931–39 283. Dorman CJ. 2007. H-NS, the genome sentinel. Nat. Rev. Microbiol. 5:157–61 284. Tisne C. 2005. Structural bases of the annealing of primer Lys tRNA to the HIV-1 viral RNA. Curr. HIV Res. 3:147–56 285. Richardson CC. 1969. Enzymes in DNA metabolism. Annu. Rev. Biochem. 38:795–840


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Contents

Annual Review of Biochemistry Volume 84, 2015


It Seems Like Only Yesterday Charles C. Richardson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 1 Veritas per structuram Stephen C. Harrison p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p37 Nuclear Organization Yosef Gruenbaum p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p61 The Balbiani Ring Story: Synthesis, Assembly, Processing, and Transport of Specific Messenger RNA–Protein Complexes Petra Björk and Lars Wieslander p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p65 Functions of Ribosomal Proteins in Assembly of Eukaryotic Ribosomes In Vivo Jesus ´ de la Cruz, Katrin Karbstein, and John L. Woolford Jr. p p p p p p p p p p p p p p p p p p p p p p p p p p p p p93 Lamins: Nuclear Intermediate Filament Proteins with Fundamental Functions in Nuclear Mechanics and Genome Regulation Yosef Gruenbaum and Roland Foisner p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 131 Regulation of Alternative Splicing Through Coupling with Transcription and Chromatin Structure Shiran Naftelberg, Ignacio E. Schor, Gil Ast, and Alberto R. Kornblihtt p p p p p p p p p p p p p p p p 165 DNA Triplet Repeat Expansion and Mismatch Repair Ravi R. Iyer, Anna Pluciennik, Marek Napierala, and Robert D. Wells p p p p p p p p p p p p p p p p 199 Nuclear ADP-Ribosylation and Its Role in Chromatin Plasticity, Cell Differentiation, and Epigenetics Michael O. Hottiger p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 227 Application of the Protein Semisynthesis Strategy to the Generation of Modified Chromatin Matthew Holt and Tom Muir p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 265 Mechanisms and Regulation of Alternative Pre-mRNA Splicing Yeon Lee and Donald C. Rio p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 291 The Clothes Make the mRNA: Past and Present Trends in mRNP Fashion Guramrit Singh, Gabriel Pratt, Gene W. Yeo, and Melissa J. Moore p p p p p p p p p p p p p p p p p p p 325 v

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Biochemical Properties and Biological Functions of FET Proteins Jacob C. Schwartz, Thomas R. Cech, and Roy R. Parker p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 355 Termination of Transcription of Short Noncoding RNAs by RNA Polymerase II Karen M. Arndt and Daniel Reines p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 381 PIWI-Interacting RNA: Its Biogenesis and Functions Yuka W. Iwasaki, Mikiko C. Siomi, and Haruhiko Siomi p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 405


The Biology of Proteostasis in Aging and Disease Johnathan Labbadia and Richard I. Morimoto p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 435 Magic Angle Spinning NMR of Proteins: High-Frequency Dynamic Nuclear Polarization and 1H Detection Yongchao Su, Loren Andreas, and Robert G. Griffin p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 465 Cryogenic Electron Microscopy and Single-Particle Analysis Dominika Elmlund and Hans Elmlund p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 499 Natural Photoreceptors as a Source of Fluorescent Proteins, Biosensors, and Optogenetic Tools Daria M. Shcherbakova, Anton A. Shemetov, Andrii A. Kaberniuk, and Vladislav V. Verkhusha p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 519 Structure, Dynamics, Assembly, and Evolution of Protein Complexes Joseph A. Marsh and Sarah A. Teichmann p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 551 Mechanisms of Methicillin Resistance in Staphylococcus aureus Sharon J. Peacock and Gavin K. Paterson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 577 Structural Biology of Bacterial Type IV Secretion Systems Vidya Chandran Darbari and Gabriel Waksman p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 603 ATP Synthase Wolfgang Junge and Nathan Nelson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 631 Structure and Energy Transfer in Photosystems of Oxygenic Photosynthesis Nathan Nelson and Wolfgang Junge p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 659 Gating Mechanisms of Voltage-Gated Proton Channels Yasushi Okamura, Yuichiro Fujiwara, and Souhei Sakata p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 685 Mechanisms of ATM Activation Tanya T. Paull p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 711 A Structural Perspective on the Regulation of the Epidermal Growth Factor Receptor Erika Kovacs, Julie Anne Zorn, Yongjian Huang, Tiago Barros, and John Kuriyan p p p 739

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Chemical Approaches to Discovery and Study of Sources and Targets of Hydrogen Peroxide Redox Signaling Through NADPH Oxidase Proteins Thomas F. Brewer, Francisco J. Garcia, Carl S. Onak, Kate S. Carroll, and Christopher J. Chang p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 765


Form Follows Function: The Importance of Endoplasmic Reticulum Shape L.M. Westrate, J.E. Lee, W.A. Prinz, and G.K. Voeltz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 791 Protein Export into Malaria Parasite–Infected Erythrocytes: Mechanisms and Functional Consequences Natalie J. Spillman, Josh R. Beck, and Daniel E. Goldberg p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 813 The Twin-Arginine Protein Translocation Pathway Ben C. Berks p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 843 Transport of Sugars Li-Qing Chen, Lily S. Cheung, Liang Feng, Widmar Tanner, and Wolf B. Frommer p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 865 A Molecular Description of Cellulose Biosynthesis Joshua T. McNamara, Jacob L.W. Morgan, and Jochen Zimmer p p p p p p p p p p p p p p p p p p p p p p p 895 Cellulose Degradation by Polysaccharide Monooxygenases William T. Beeson, Van V. Vu, Elise A. Span, Christopher M. Phillips, and Michael A. Marletta p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 923 Physiology, Biomechanics, and Biomimetics of Hagfish Slime Douglas S. Fudge, Sarah Schorno, and Shannon Ferraro p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 947 Indexes Cumulative Index of Contributing Authors, Volumes 80–84 p p p p p p p p p p p p p p p p p p p p p p p p p p p 969 Cumulative Index of Article Titles, Volumes 80–84 p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 973 Errata An online log of corrections to Annual Review of Biochemistry articles may be found at http://www.annualreviews.org/errata/biochem

Contents

vii

SIRT3: as simple as it seems?

Stimulation is never quite as simple as it seems.

Measuring visual acuity is not as simple as it seems.

Not What It Seems: Deep Tissue Infection Presenting as Cellulitis.

Larvae in a traveller's faeces. Not always what it seems.

Dementia: Mild cognitive impairment--not always what it seems.

It Seems Like the Resuscitation Happened Ages Ago-Chronic Critical Illness in Children: Digging in for the Long Haul.

Telling it like it is.

RNA: yesterday, today and tomorrow.

Neurosyphilis yesterday and today.

Submucosal lesion of the oesophagus: not everything is what it seems.

Hemiparesis in an Adolescent With Acute Lymphoblastic Leukemia: Everything Is Not Always What it Seems.

Not paying for catheter-associated urinary tract infections: more difficult than it seems?

Headache yesterday in Europe.

Dendritic cell subset composition in the human liver is more complex than it seems.

It saddens me that race seems to have dropped off the agenda.

Can super-duper centenarians possibly matter? Increasingly it seems they may.

The aerodynamic cost of head morphology in bats: maybe not as bad as it seems.

Solar protection by hair: more complex than it seems to be.

The lingering effects of testosterone abuse - it seems muscles have long memories.

"It just seems outside my health": How Patients with Chronic Conditions Perceive Communication Boundaries with Providers.

Reply to: "Dendritic cell subset composition in the human liver is more complex than it seems".

Apolipoprotein L2 contains a BH3-like domain but it does not behave as a BH3-only protein.

Posterior iliac crescent fracture-dislocation: is it only rotationally unstable?