The Chromosome Queen

Credit: JASON VARNEY | varneyphoto.com Nancy Kleckner became smitten with genetic material as a high school student during the early 1960's, in the dawning of molecular genetic research. "DNA is intrinsically interesting," says Kleckner, now a professor in Harvard's department of molecular and cellular biology (MCB). In addition to being the basis of all life on earth, she says DNA "has a pleasing aesthetic quality to it in terms of how the molecule is designed." As an undergraduate at Harvard, Kleckner worked with Matt Meselson in a department that was progenitor of MCB. While there, she learned how to think about science. "For me, the most important thing you learn from the people with whom you study is how they think about science," she says. "And Matt is high on the list of people who think about science in really interesting ways." Kleckner took that lesson to heart. "She's extremely bright and extremely innovative," says Gareth Jones of the University of Birmingham. Job Dekker of the University of Massachusetts Medical School, and Kleckner's former postdoc, agrees. "Her originality is quite amazing," he says. "She comes up with ideas and you think, ?I don't see how she got there. What's the logic between the facts that we see in front of us and arriving at this hypothesis?' She has some kind of scientific intuition that makes her see things in ways different from how other people see them. And it turns out, she's usually right." Kleckner has used that originality and intuition to make great strides in addressing questions about chromosome behavior, including the movement of transposons, the regulation of bacterial replication, and the interaction of homologs during meiosis. "She has the career of a first-class scientist," says David Botstein of Princeton University, Kleckner's postdoctoral mentor. "Over and over again, whatever problem she decides is worth her time, she makes a serious impact on. And that's just what you'd expect from a ?Top-1%' kind of scientist." CUT-AND-PASTE TRANSPOSITION After graduating from Harvard in 1968, Kleckner earned her PhD at Massachusetts Institute of Technology working with Ethan Signer on the genetics of lambda phage and DNA replication. She then joined Botstein's group as a postdoc in 1974, around the time that he and his lab were beginning to believe that a segment of DNA that encoded tetracycline resistance was able to jump around from one place to another, she says. "Of course everyone knew about Barbara McClintock's work," Kleckner notes. "But this was the first indication that antibiotic resistance genes could be carried on what are now called transposable elements. And my first contribution as a postdoc was to show definitively that that was true." Kleckner also showed that the transposon in question, Tn10, moved by excising itself from one location and pasting itself in another. She purified the elements responsible for this cut-and-paste mode of transportation, learned how they worked, and then labored to turn the system into a tool for mapping bacterial genes and making mutations. By 1975, she and her colleagues had published the first report on transposon mutagenesis. "I've had occasion to look at that paper recently," says Botstein. "And it still strikes me as a terrific paper, because it's the whole story. And it's all still true. Not everybody gets to have a paper like that." The system "worked like a charm," says Susan Gottesman from the National Institute of Health. As a fellow postdoc from Botstein's lab, Gottesman used Kleckner's constructs to generate Escherichia coli mutants. "Those mutants we made, we still use." LARGER NUMBER OF ANGSTROMS In 1977, Kleckner returned to Harvard as a junior faculty member. Although she would continue to work on transposons for almost another 20 years, she decided she wanted to broaden her interests. "The problems in the transposition field were becoming more and more particular and more and more about issues at small numbers of angstroms," she says. "I wanted to keep working at larger numbers of angstroms." Receiving tenure in 1985 gave her the breathing room she needed to expand her pursuits, including the study of bacterial replication. "I grew up in the tradition of early molecular biology in which E. coli and Salmonella and bacterial viruses were small simple organisms you could use to understand the basic principles of life." At the time, she says, people were pondering how bacteria could replicate and segregate their chromosomes as they divide "without producing a mess." Specifically, the cells need a mechanism to ensure that they copy each chromosome once, and only once, each time they reproduce. Kleckner modeled the process and discovered, among other things, SeqA, a protein that prevents replication from being initiated more than once on any chromosome. "She was the first, I believe, to think there must be a negative factor limiting initiation to once per cell cycle," says Eric Boye of the Norwegian Radium Hospital in Oslo. "And she went after this hypothetical factor and found it." She then went on, in collaboration with Boye and others, to determine that SeqA works by recognizing and binding to the hemimethylated sequences that signal newly replicated DNA - work that was published in 1994. THE BABY CELL COLUMN Kleckner continues to explore how replicated chromosomes physically separate and move into daughter cells. To do this, she and her lab have developed a "baby-cell column" that allows them to isolate "newborn" bacteria that are all at the same stage of the division process. The column is packed with beads on which bacteria are suspended by their flagella. Newborn cells lack flagella and fall through the column, where they can be collected and examined. Previous methods for isolated such "synchronized" bacteria were limited in use or somewhat disruptive to the cells, says Gottesman. "Nancy's lab made the process much more elegant and much more effective." "She's always willing to invest the time and energy it takes to develop new methods, rather than simply asking the questions that can be asked given the methodologies that are currently available." --Doug Bishop Her penchant for elegant techniques runs throughout her research. "She never lets technical hurdles influence her decision about what she wants to work on," says former postdoc Doug Bishop of the University of Chicago. "She's always willing to invest the time and energy it takes to develop new methods, rather than simply asking the questions that can be asked given the methodologies that are currently available." New techniques were key to the advances Kleckner made in her studies of mechanisms underlying meiosis and recombination, the third major area of research that she and her lab pursue. "At the time we started, there was no molecular anything about meiosis, roughly speaking," says Kleckner. "So we started by trying to work out some genetics and some physical assays for the events of recombination." Among other things, she and her lab tamed a wild isolate of yeast that underwent meiosis more quickly and efficiently than existing laboratory strains. They developed novel methods for mapping the location of proteins on whole chromosomes, isolating recombination intermediates, and examining chromosome structure. The latter took Dekker four years to establish - an example of Kleckner's commitment to "the development of technologies that will allow you to learn entirely new things," he says. Out of those techniques came many discoveries, including the enzyme that generates the double-strand breaks that kick off recombination, the recombinase that promotes the alignment of the resulting "broken" DNA with its matching sequence on the homologous chromosome, and the formation of side-by-side Holliday junctions. But perhaps most importantly, she proved that the double-strand breaks that initiate recombination happen early, before the formation of the synaptonemal complex - the protein-coated interface that brings homologs into intimate juxtaposition. Her findings not only "revitalized meiosis," says Scott Hawley of the Stowers Institute in Kansas City, Mo., but also "basically turned the whole view of the process on its head." "The paradigm up until Nancy had been: You paired chromosomes, you built a synaptonemal complex between them, and then you recombined them," says Hawley. "The idea that in yeast you needed double-strand breaks to build the synaptonemal complex was really quite shocking." "Her contributions to meiosis can't be overstated," says Jones. And she's not through yet. "With Nancy, you're always on to the next question. There isn't a lot of resting," says Bishop. "You move from having to do more work to make sure your interpretation is correct, on to the next thing without a point at which everyone stops and opens a bottle of champagne." When it comes to her science, he says, Kleckner is relentless and intense. "Her demeanor is as if she was just put in charge of making sure the Apollo 13 astronauts get back to earth safely. There's always this sense of importance and urgency." CROSSOVER RESEARCH Kleckner's current mission is an attempt to use principles of physics and engineering to deconstruct a phenomenon known as crossover interference. When homologs align during meiosis, they exchange genetic material. The sites of these exchanges, called crossovers, tend to be evenly spaced along the chromosomes. Kleckner says she thinks this happens because chromosomes breathe. "If you have two chromatin masses, they might expand and push against each other," she says. "That will produce compression along the axes of the chromosomes and at the interfaces between them." At a certain point, the stress will cause one of the bridges between the chromosomes to buckle, producing a crossover. Once that happens, the stress in that region is relieved, so no further crossovers will occur nearby. "It's an original and innovative way of thinking about how chromosomes behave," says Jones. "What's interesting is, if you go back 60 or 70 years, people interested in meiosis did tend to think about things in mechanical terms, but that got sort of subsumed by the growth of molecular biology and biochemistry. But I think people are coming back to realize that physical factors like mechanical stress acting on chromosomes and the molecules within them could be very important. And Nancy's largely responsible for developing this idea in recent years." "It might be right, or it might be wrong," says Kleckner. "But its importance for my own research is that it made me appreciate that if you started to look at chromosomal phenomena from a physics or engineering perspective, you ask different questions and might do different experiments. It gets you away from a simple molecular description of stuff and it gets you back to invoking basic principles of physics, which is of course where molecular biology started." And chances are she's right. "Some people feel she's visionary, others feel she's too speculative," says Bishop. "But if you keep track of how many of her ideas turn out to be correct, it's impressive." Dekker agrees: "To be honest, I can't think of any examples where her intuition turned out to be wrong." She's even right when it comes to other people's work. After Hawley gave a presentation at a meeting in the Swiss Alps, he says, "Nancy sat down with me and proposed this completely different hypothesis." A few years later, he discovered that "Nancy was entirely correct. Things were exactly as she had predicted." "Embarrassing as that is," laughs Hawley, "one of most exciting things about Nancy is that she's virtually always right." khopkin@the-scientist.com

The Chromosome Queen

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Karen Hopkin

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April 2007

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