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Home , Jean Weigle, Matthew Meselson

LASKER SPECIAL ACHIEVEMENT IN MEDICAL SCIENCE AWARD COMMENTARY

Explorations in the land of DNA and beyond

Matthew Meselson

Like many of my colleagues, I have liked sci- the war was still evident and the must have had up his sleeve all along: the X- ence for as long as I can remember. Though was starting up. ray diffraction determination of the structure not college educated, my parents were always The following year, with almost no under- of N,N´-dimethylmalonamide. The idea was encouraging, as were two uncles who knew a graduate science credits, I entered the to test the prediction from his resonance the- little chemistry. When I was still in grammar California Institute of Technology, a fresh- ory that its two amide groups are planar, as of school, the basement of our house in Los man all over again. I took ’s course they turned out to be. http://www.nature.com/naturemedicine Angeles became a sizeable laboratory where I general chemistry course and did a research I remember being disappointed at the time built radios, spectroscopes and other instru- project for him to determine the accessibility by Erwin Schrödinger’s little 1945 book, ments, purified radium and rare-earth ele- of the heme group of the of the What Is Life?1.A founder of quantum ments from carnotite and monazite ores by marine worm Urechis to a series of increas- mechanics, Schrödinger had left his native fractional crystallization and ion-exchange ingly large alkyl isocyanides that I synthe- Austria soon after the German occupation in chromatography, and synthesized for neigh- sized. When at the end of the year I showed 1938 and had become a professor at Trinity bors the recently discovered insecticide DDT. Pauling my only partly complete results, College, Dublin. After pointing out that heat The war, though far away, was neverthe- expecting a stern response, he beamed his motion would make assemblages of inde- less the focus of attention then, and it broad smile and said that the important pendent molecules too unstable to account seemed wrong to spend high school vaca- thing for a student to learn from such work for the stability of , he concluded that tions solely in recreational activities. So I was that it can take much longer to complete genes, or maybe even entire chromosomes, took a job washing glassware at a vitamin than expected. are huge molecules, the stability of which factory and attended summer school classes I was dissatisfied with undergraduate life derives from their covalent bonds, and that

© 2004 Nature Publishing Group to shorten the time required to earn a at Cal Tech. Except for general chemistry, the their variety corresponds to their various sta- diploma and begin college. To my surprise, courses I took seemed based too much on ble isomeric states. Though couched in quan- however, academic credits were not enough: memory and, after Chicago and half a year in tum-mechanical terms, the concept boiled California law required 3 full years of high Europe, the other students (all males and 4–5 down to what for chemists is a truism—that school physical education. Then I learned of years younger) seemed uninterested in the only covalent bonds can account for the sta- a college that took students without requir- world outside. Eventually, after a year back at bility of the hereditary substance. Yet coming ing the last 2 years of high school—the Chicago taking chemistry and math courses from Schrödinger and written so gracefully, . and a year at Berkeley as a physics graduate with a moving epilog on determinism and Upon entering the College of the student, through great good luck I became free will, it influenced some of the pioneers of University of Chicago in 1946, expecting to Linus Pauling’s last graduate student at Cal molecular biology to enter the field. study chemistry and physics, I found that Tech.Initially, he suggested that for my dis- Schrödinger addressed the question of the undergraduate programs in specialized sub- sertation research I should determine the chemical stability of the genetic material but jects had been abolished in favor of a manda- structures of some tellurium minerals by said nothing about how it might replicate. It tory curriculum based on classical writings in means of X-ray diffraction. He may have was Pauling, in 1948, who grasped the the humanities and the social and natural sci- been only half-serious, because he went on to answer, published at the University of ences. I count this elementary education in caution me that some chemists working with Nottingham as the 21st Sir Jesse Boot what is uninformatively called the ‘liberal tellurium compounds had acquired a horri- Foundation Lecture entitled “Molecular arts’ a piece of good fortune, even though it ble halitosis called ‘tellurium breath,’ isolat- Architecture and the Processes of Life”: got me only a Ph.B. (bachelor of philosophy), ing them from society and driving some of no good at all for admission to a graduate them to suicide. By then wondering how In general, the use of a or virus as a school in science. After 3 years, I left Chicago ordinary atoms could be put together to template would lead to the formation of a and spent the next 6 months traveling make self-replicating structures, I asked to be molecule not with identical structure but around Europe, reading and discussing with assigned instead a molecule composed of with complementary structure. It might hap- friends what might lie ahead for America and biologically more important atoms. So pen, of course, that a molecule could be at the the world. It was 1949—the devastation of Pauling suggested another project that he same time identical with and complementary

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tern.He interpreted the result to mean that and shouted, “The most important develop- the molecule consisted of three chains coiled ment in biology in a decade and you don’t about one another. The calculation appears know about it? Read these and don’t come on a page dated 26 November 1952 in one of back until you have!” I took this as an his research notebooks, followed by the invitation to come back. When I did, Max notation, “Perhaps we have a triple-chain spoke of his difficulty in imagining that the structure!” Of course, the three-chain struc- two polynucleotide chains wound around ture he and Corey published three months each other could come apart during replica- later was wrong. Soon after receiving from tion without breaking—an understandable and a prepubli- dilemma, because DNA topoisomerases were cation copy of their April 1953 letter to then unknown. How this question led to the Nature describing their double-helical invention of equilibrium density-gradient model of DNA, Pauling wrote to his son centrifugation that formed the second half of Peter, “The structure seems to me to be a my doctoral dissertation, and to the experi- very interesting one and I have no strong ment that Frank Stahl and I then did using argument against it. I do not think their 15N as a density label, demonstrating the arguments against our structure are strong semiconservative replication of DNA, is told ones, either.” He went on to write that if the in detail in a book by Frederick Lawrence specimens from which his data came con- Holmes (Fig. 1)2–4. tained about 30% water, the DNA molecules Density-gradient centrifugation proved in them would have only two chains. useful for attacking other fundamental prob- Figure 1 and Frank Stahl in My first knowledge of the great discovery lems posed by the double-helical structure of 1996, standing at the place they met in 1954 at of Watson and Crick came through an act of DNA. and I used it in 1960 to http://www.nature.com/naturemedicine Woods Hole. From ref 2. violence. Sometime in 1953, after the publi- show that genetically recombinant chromo- cation of the double helix, I went for the first somes of phage lambda contain segments of to the template on which it is moulded. time to talk with Max Delbrück. Immediately parental DNA consistent with a break-and- However, this case seems to me to be too he asked what I thought of the papers Jim join model of recombination rather than unlikely to be valid in general, except in the and Francis had published in Nature in April with the template-switching model called following way. If the structure that serves as a and May. When I replied that I had not heard copy choice5. Earlier that year, Cedric Davern template (the gene or virus molecule) consists of them, Max hurled a stack of reprints at me and I showed by a density-transfer experi- of, say,two parts, which are themselves com- plementary in structure, then each of these parts can serve as the mould for the produc- tion of a replica of the other part, and the complex of two complementary parts thus can serve as the mould for the production of

© 2004 Nature Publishing Group duplicates of itself.

Pauling attributed the templating speci- ficity involved in gene duplication, as well as in certain other manifestations of biological specificity, to detailed structural complemen- tarity and the cooperative action of individu- ally rather weak interactions, such as van der Waals attraction and hydrogen bonding. Later, it was learned that the great fidelity of gene duplication is achieved by the repeated application of such interactions in certain error-correction mechanisms. Despite Pauling’s insight requiring the gene to be made of two complementary parts, he proposed a three-strand structure for DNA (and RNA). From an X-ray diffrac- tion measurement of the spacing between DNA molecules in a DNA fiber (assuming a hexagonal lattice), the fiber’s density, and the nucleotide molecular weight (but making no allowance for the contribution of water), he calculated the distance per residue along the molecule to be almost exactly one-third of Figure 2 François Jacob, Max Delbrück, Matthew Meselson, Ronald Rolfe (partly hidden from view), the 3.4-Å repeat seen in the diffraction pat- Gunther Stent and in Pasadena, June 1960. From ref. 16.

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ment that ribosomal RNA molecules of Upon moving to Harvard in 1961, I con- mental problem in biology. Although the are stable for many genera- tinued to study mechanisms of genetic that accompanies tions6.Achieving the necessary resolution for recombination8 and later worked on host- sexual reproduction can generate variants of both of these experiments required greater controlled restriction and modification of increased fitness, it also can tear apart well- density labeling than could be achieved with DNA and on DNA mismatch repair, leading adapted gene combinations, and it imposes 15N alone and became possible only with the to the prediction and then the demonstra- various other costs, including those of pro- availability of isotopically pure 13C. This was tion, with Miro Radman, of methyl-directed ducing and maintaining males. A possible made by isotope diffusion in Moscow specifi- DNA mismatch repair9,10.In attempting to experimental approach to the problem is the cally for me at the request of Pauling. It was isolate an activity responsible for DNA study of rotifers of the class Bdelloidea, a essential also for the experiment conceived restriction in E. coli K12, I discovered an ATP highly unusual group of diminutive freshwa- by Sydney Brenner, who came to Cal Tech requirement for degradation of unmodified ter invertebrates of worldwide distribution with François Jacob to join with me for a few DNA in extracts of restricting cells. Attempts comprising some 370 described species in weeks in 1960 to test the messenger hypothe- at purification led to loss of activity, how- which, despite much study, males, hermaph- sis of Jacob and Monod (Fig. 2). ever. At that point, Bob Yuan came to our lab rodites and meiosis are unknown, and which It was known that T-even phage infection and joined me in attempting purification of has nevertheless persisted and successfully of E. coli brings about the synthesis of a the enzyme. From unpublished observations evolved for tens of millions of years. Bdelloid short-lived RNA with base composition like of Bill Wood on the effect of methionine eggs are produced from oocytes by two that of the phage, shuts down most other deprivation on restriction, we got the idea mitotic divisions, without chromosome RNA synthesis and diverts amino acid incor- that methionine might also be required. By pairing and without reduction in chromo- poration to the massive synthesis of phage adding both ATP and methionine we could some number—each oocyte yielding one egg proteins. The messenger hypothesis pre- continue the purification, but the activity and two polar bodies. Since I first learned of dicted that the short-lived phagelike RNA soon decreased again. At that point, Bob these creatures in 1989, we have conducted a and the newly synthesized phage proteins realized that S-adenosylmethyltransferase in series of experiments to provide molecular http://www.nature.com/naturemedicine would be found on ribosomes that were the partially purified preparation might be genetic tests of their ancient asexuality and already present before phage infection. The making S-adenylmethionine (SAM) from to explore what may have allowed them to protocol was to infect bacteria grown in 13C methionine and ATP. At first, we thought dispense with sex without going extinct13–15. 15N heavy-isotope medium with phage T4, that only SAM would be needed, but we One of the several unusual characteristics we immediately transfer the culture to light-iso- soon found that SAM and ATP are both have discovered is their apparent lack of tope medium containing 32P or 35S to label needed, allowing us to purify the endonucle- retrotransposons, mobile genetic elements RNA or protein, and then determine the dis- ase to homogeneity11,12.We discovered to that are found in nearly all of the numerous tribution of the radioisotopes after CsCl den- our surprise that, even in the limit digest, not and diverse eukaryotes in which they have sity-gradient centrifugation. The position of all lambda molecules are broken at the same been sought. Such elements are generally ribosomes was determined by their UV sites. The argument followed from the sedi- deleterious, and one class of them, the non- adsorption. François, suffering from World mentation distribution of the digested LTR (non–long terminal repeat) retrotrans- War II shrapnel wounds in his legs, kept over- lambda pieces. For example, although the posons, seems to be as ancient as meiosis all watch on the proceedings. I mainly ran the size of some pieces was in the range of 40% itself. Transposable elements are capable of

© 2004 Nature Publishing Group centrifuge, and Sydney worked furiously not of a lambda chromosome, they accounted autonomous increase within the genomes only on the messenger experiment but also for less than 40 mass % of the digest. This they inhabit, but their number can be kept in on helping me late at night to score phage lack of site specificity, alas, made EcoK and check by mechanisms whose effectiveness plaques from my lambda recombination the other type I endonucleases we studied increases disproportionately as their copy experiments. The chief obstacle we had to useless for genetic engineering. We also number increases. In sexual species, this can overcome was the instability of ribosomes in found that the enzyme nicks before it makes occur by purifying selection through ectopic the CsCl density gradients. Only in the last a double-strand break and that hybrid DNA crossing-over, leading to inviable aneuploi- few days of their planned stay, after I had to made by annealing modified and unmodi- dies, and by homologous recombination if leave on a trip east, did Sydney find that fied strands is neither nicked nor cleaved, the deleterious effects of insertions interact greatly increasing the magnesium ion con- meaning that the enzyme looks at both synergistically. Because asexual organisms centration would stabilize the particles and chains before deciding what to do, providing lack both of these mechanisms and would make the experiment work. The newly made a basis for protecting a cell from its own also lack any other control mechanisms that RNA (and protein, as later found by Sydney restriction system. ’s Nobel depend on meiosis, we have wondered in Cambridge) were found to be associated account of his outstanding work on restric- whether an important cause of the early with the heavy ribosomes, just as predicted7. tion and modification acknowledges our extinction of asexual organisms and, corre- The experiment I had done with Davern had work on EcoK as having set the basis for his spondingly, of the relative advantage of sex- left the possibility that ribosomal RNA could in vitro studies of these processes. ual reproduction is the ability of sex to be informational, even though there was evi- Sometime around 1970 I read an article combat the unchecked increase of deleteri- dence that at least some informational RNA, that asked not how genetic recombination ous retrotransposons. If so, much evolu- particularly that for β-galactosidase, was works but why it exists. There are many tionary theory would need to be short-lived. Finding phage RNA and phage hypotheses but no general agreement about re-examined, providing new insights into protein on ribosomes that had been made what makes sexual reproduction so nearly many issues, among them the causes of before there was any phage information in universal in animals and plants, and what extinction of asexual lineages and, perhaps, the cell effectively ruled this out and con- causes the relatively early extinction of even that of some species that reproduce firmed the messenger hypothesis. nearly all lineages that abandon it—a funda- sexually.

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1. Schrödinger, E. What Is Life? (Macmillan, New York, somes for protein synthesis. Nature 190, 576–581 tion or genetic exchange. Science 288, 1211–1215 1945). (1961). (2000). 2. Holmes, L.F. Meselson, Stahl, and the Replication of 8. Meselson, M. The molecular basis of genetic recom- 14. Mark Welch, J.L., Mark Welch, D.B. & Meselson, M. DNA (Yale University Press, New Haven, 2001). bination in Heritage from Mendel (ed. Brink, R.A.) Cytogenetic evidence for asexual of 3. Meselson, M., Stahl, F. & Vinograd, J. Equilibrium 81–104 (University of Wisconsin Press, Madison, Bdelloid rotifers. Proc. Natl. Acad. Sci. USA 101, sedimentation of macromolecules in density gradi- 1967). 1618–1621 (2004). ents. Proc. Natl. Acad. Sci. USA 43, 581–588 9. Wagner, R. & Meselson, M. Repair tracts in mismatch 15. Arkhipova, I. & Meselson, M. Transposable elements (1957). DNA heteroduplexes. Proc. Natl. Acad. Sci. USA 73, in sexual and ancient asexual taxa. Proc. Natl. Acad. 4. Meselson, M. & Stahl, F. The replication of DNA in E. 4135–4139 (1976). Sci. USA 97, 14473–14477 (2000). coli. Proc. Natl. Acad. Sci. USA 44, 671–682 10. Meselson, M. Methyl-directed repair of DNA mis- 16. Judson, H.F. The Eighth Day of Creation (Cold Spring (1958). matches in The Recombination of Genetic Material Harbor Laboratory, New York, 1996). 5. Meselson, M. & Weigle, J. Chromosome breakage (ed. Low, K.B.) 91–113 (Academic Press, San Diego, accompanying genetic recombination in bacterio- 1988). phage. Proc. Natl. Acad. Sci. USA 47, 857–868 11. Meselson, M. & Yuan, R. DNA (1961). from E. coli. Nature, 217, 1110–1114 (1968). Matthew Meselson is in the Department of 6. Davern, C.I. & Meselson, M. The molecular conserva- 12. Meselson, M., Yuan, R. & Heywood, J. Restriction Molecular and Cellular Biology, Harvard tion of RNA during bacterial growth. J. Mol. Biol. 2, and modification of DNA. Annu. Rev. Biochem. 41, University, 7 Divinity Avenue, Cambridge, 153–160 (1960). 447–466 (1972). 7. Brenner, S., Jacob, F. & Meselson, M. An unstable 13. Mark Welch, D.B. & Meselson, M. Evidence for the Massachusetts 02138, USA. intermediate carrying information from genes to ribo- evolution of Bdelloid rotifers without sexual reproduc- e-mail: [email protected] http://www.nature.com/naturemedicine © 2004 Nature Publishing Group

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