How DNA Replicates

How DNA Replicates By Matthew Meselson and Frank Stahl A Key Experiment produced by The Explorer’s Guide to Biology 2 Th e Explorer’s Guide to Biology https://explorebiology.org/ How DNA Replicates Matthew Meselson, Harvard University, and Franklin W. Stahl, University of Oregon Matthew Meselson Matthew Meselson had a passion for physics and chemistry throughout his early life, often conducting science experiments in his family’s garage. At the age of 16, he enrolled at the University of Chicago, beginning an academic career that led to doctoral studie s at the California Institute of Technology under Linus Pauling. In addition to his widely known work demonstrating semi-conservative replication of DNA with Frank Stahl, Meselson has made many key discoveries in the molecular biology. He is also known for his work in limiting the proliferation of chemical and biological weapons. Meselson is a member of the National Academy of Science and a recipient of the Lasker Award. He continues to serve as a member of the faculty at Harvard University, where he has taught and conducted research since 1960. Franklin Stahl Following a sheltered life in a Boston suburb (Needham), Frank stum- bled his way through college (Harvard, 1951) before fl eeing to a graduate school in biology (U Rochester) to avoid the military draft. While in the graduate school, Frank took a course taught by A. H. (Gus) Doermann, and, for the fi rst time in his life, he had a goal. With Gus, he studied genetic recombination in phage. To meet a departmental requirement, Frank took a summer course at Woods Hole, where he met Matt Meselson and began the work described in this Key Experiment. In 1959, Frank joined the faculty at the University of Oregon, Eugene, in their new Institute of Molecular Biology. He has been there ever since. Frank is now an emeritus faculty member who still enjoys teaching as well as family life and the natural wonders of Oregon. 3 How DNA Replicates Summary Some experiments have proven so influential that they have been christened with the names of the scientists who performed them. The “Meselson–Stahl experiment” is one of those. It has also been called “the most beautiful experiment in biology,” a title that has seemed to stick over the years. Why was the Meselson and Stahl experiment so important? Their experiment provided the first critical test of the Watson–Crick models for the structure of DNA and its replication, which were not universally accepted at the time. The convincing results of the Meselson–Stahl experiment, however, dispelled all doubts. DNA was no longer just an imaginary model; it was a real molecule, and its replication could be followed in the form of visually compelling bands in an ultracentrifuge. Meselson and Stahl found that these DNA bands behaved in the ultracentrifuge exactly as Watson and Crick postulated they should. Why was the Meselson–Stahl experiment “beautiful”? Because it was conceptually simple and yet sufficiently powerful to differentiate between several competing hypotheses for how DNA might replicate. Taken together, the Watson–Crick model and the Meselson–Stahl experiment marked the transition to the modern era of molecular biology, a turning point as impactful as the theory of evolution. The story of the Meselson–Stahl experiment, as told here by its protagonists, also reveals how friendship and overcoming obstacles are as cru- cial to the scientific process as ideas themselves. Learning Overview Big Concepts Faithful replication of the genetic material (DNA) is the foundation of all life on earth. The experiment by Meselson and Stahl established that DNA replicates through a semi-conservative mech- anism, as predicted by Watson and Crick, in which each strand of the double helix acts as a template for a new strand with which it remains associated, until the next replication. Terms and Concepts Used Bacteriophage, bases, base pairing, chromosome, DNA, Hershey–Chase experiment, eukaryote, mutations, nucleotides, phage, prokaryote, recombination, RNA, ultraviolet light Terms and Concepts Explained Equilibrium density-gradient centrifugation, DNA replication, isotope, semi-conservative DNA replication 4 How DNA Replicates Introduction Matthew Meselson and Franklin Stahl (both 24 years old) met at the Marine Biological Laboratory in Woods Hole in Massachusetts and decided to test the Watson–Crick model for DNA replication, which was unproven at the time. What Events Preceded the Experiment? Watson and Crick proposed a “Semi-Conservative” model for DNA replication in 1953, which derived from their model of the DNA double helix. In this proposal, the strands of the duplex separate and each strand serves as a template for the synthesis of a new complementary strand. Watson’s and Crick’s idea for DNA replication was a model, and they did not have data to support it. Some prominent scientists had doubts. Two other models, “Conservative” and “Dispersive”, for DNA replication were proposed. Setting Up the Experiment A method was needed to detect a difference between the parental and daughter (newly replicated) DNA strands. Then, one could follow the parent DNA molecule in the progeny. Meselson thought to distinguish between parental and newly synthesized DNA using a density difference in the building blocks (nucleotides) used to construct the DNA. The three models for DNA replication would predict different outcomes for the density of the replicated DNA in the first- and second-generation daughter cells. The general experimental idea was first to grow bacteria in a chemical medium to make high-density DNA and then abruptly shift the bacteria to a low-density medium so that the bacteria would now synthesize lower density DNA during upcoming rounds of replication. The old and newly synthesized DNA would be distinguished by their density. To measure a density difference in the DNA, Meselson and Stahl invented a method called equilibrium density gradient centrifugation. In this method, the DNA is centrifuged in a tube with a solution of cesium chloride. When centrifuged, the cesium chloride, being denser than water, forms a density gradient, reaching a stable equilibrium after a few hours. The DNA migrates to a point in the gradient where its density matches the density of the CsCl solution. Heavy and light DNA would come to different resting points and thus physically separated. Doing the Key Experiment Meselson and Stahl first decided to study the replication of DNA from a bacteriophage, a virus that replicates inside of bacteria, and used a density difference between two forms of the nucleo- base thymine (normal thymine and 5-bromouracil). These experiments did not work. 5 How DNA Replicates The investigators changed their plans. They studied replication of the bacterial genome and used two isotopes of nitrogen (15N (heavy) and 14N (light)) to mark the parental and newly synthesized DNA. When the population of bacteria doubled, Meselson and Stahl noted that the DNA was of an intermediate density, half-way between the dense and light DNA in the gradient. After two dou- blings, half of the DNA was fully light and the other half was of intermediate density. These results were predicted by the Semi-Conservative Model and are inconsistent with the Conservative and Dispersive Models. Meselson and Stahl did another experiment in which they used heat to separate the two strands of the daughter DNA after one round of replication. They found that one strand was all heavy DNA and the other all light. This result was consistent with the Semi-Conservative model and provided additional evidence against the Dispersive Model. Overall, the results provided proof of Semi-Conservative replication, consistent with the model proposed by Watson and Crick. What Happened Next? Within a couple of weeks after their key experiment, Meselson wrote a letter to Jim Watson to share news of their result (letter included). Max Delbruck, the Caltech physicist and biologist who had proposed the dispersive model, was elated by the results, even though Meselson and Stahl disproved his replication hypothesis, and urged the young scientists to write up their results for publication and announce the important result to the world (1958). Scientists now know a great deal about the protein machinery responsible for DNA replication. Closing Thoughts The Meselson–Stahl experiment had a powerful psychological effect on the field of genetics and molecular biology. It was the first experimental test of the Watson and Crick model, and the results clearly showed that DNA was behaving in cells exactly as Watson and Crick predicted. In addition to having a good idea, the behind-the-scenes tour of the Meselson–Stahl experiment reveals that friendship and persistence in overcoming initial failures play important roles in the scientific discovery process. Also important was an atmosphere of freedom that allowed Meselson and Stahl, then very junior, to pursue their own ideas. Guided Paper Meselson, M. and Stahl, F.W. (1958). The replication of DNA in Escherichia coli. Proceedings of the National Academy of Sciences U.S.A., 44: 672–682. 6 How DNA Replicates Introduction The first conversation between Matt Meselson and Frank Stahl, in the summer of 1954, began a collaboration that led to their Key Experiment on DNA replication and marked the beginning of a lifelong friendship. Matt and Frank describe the circumstances that brought them together below. Matt It was 1954, the year after Jim Watson and Francis Crick published their two great papers describ- ing their double helical model of DNA and its implications for how it might replicate, mutate, and carry genetic information. Jim Watson (26 years old) and I (a 24-year-old first-year graduate student) were both at Caltech and living at the Athenaeum, the Caltech faculty club. We often talked while waiting for dinner.

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