Crossing Over

Bishop subsequently slowed his pace—at least with regards to his sleep-wake cycle. But he didn’t lose his momentum, and by the time he’d done his PhD and a postdoc, Bishop had racked up a handful of papers in Nature, Science, and Cell. As a graduate student, he examined mismatch repair in yeast—the subject of his first Nature paper—but his subsequent publications have focused on the molecular mechanisms that drive homologous recombination during meiosis. During his postdoc, Bishop discovered Dmc1—an enzyme that plays a central role in meiotic recombination in yeast. And he continues to study how Dmc1, and its relative Rad51, work together to catalyze the strand exchange reaction that lies at the heart of homologous recombination.

“Doug manages to integrate cytological, molecular, and genetic approaches to studying meiotic recombination,” says Michael Lichten of the National Cancer Institute. “His studies have shaped the way we approach the study of recombination in meiosis.”

Bishop’s interest in meiosis stretches back to his grad school days at Harvard. “Rich Losick was studying sporulation in Bacillus —doing really beautiful science,” Bishop says. “And I knew that yeast also make spores. So I wanted to do something similar in yeast.” Geneticist Helen Greer agreed to let him try, but she then had a change of heart—about her own life in science. “My advisor decided she didn’t want to be a biologist anymore,” says Bishop. “So when she left for law school, we all had to relocate.”

Richard Kolodner, who was then at Harvard, took Bishop in. “I figured that someone in his position probably felt he had something to prove,” he says. And Bishop did not disappoint. “He was working on DNA mismatch repair, which had been well characterized in bacteria,” says Kolodner. But people had begun to question whether eukaryotes had a similar mechanism for correcting single-nucleotide mistakes. “What Doug did is test directly whether yeast had mismatch repair. And he showed they did,” says Kolodner, now at the UC San Diego School of Medicine. “So his work is pretty pivotal in the field of mismatch repair in higher eukaryotes.”

But meiosis still called to him. So when Bishop was seeking a postdoctoral position in 1988, rumors that Harvard’s Nancy Kleckner was about to launch a project on meiosis drew him to her lab. There he decided that he would try to identify key components of the synaptonemal complex—the zipperlike protein structure that forms along the length of paired homologous chromosomes—by looking for genes that are turned on during meiosis. “It was like a functional genomics project before anyone called it that,” says Bishop. “I set out to make a library of clones that represented genes that are only expressed during meiosis.”

“But instead of stopping there,” says Kleckner, “Doug did something smarter.” Kleckner was famed for her work using the transposon Tn10 to interrupt genes. So Bishop decided he would take his collection of meiosis-specific clones and mutagenize them with Tn10. He could then introduce those pieces of transposon-disrupted DNA into yeast and specifically knock out every meiotically induced gene.

“It was going to be great,” says Bishop. But working on such a large scale was no walk in the park. His library, he says, was not quite up to snuff. And getting all the insertions in the right place was tricky. Finally, in an effort to test the system, Bishop selected one clone from his library at random. He inserted the transposon, allowed the yeast cells’ general recombination machinery to swap the interrupted gene for the native copy, and found that the mutation shut down meiosis.

“Once we had that phenotype,” Bishop says, “we thought, well, let’s go ahead and look at the sequence [of the gene].” That way they could maybe get a handle on what it is they’d cloned. A few gels later, Bishop typed his nucleotide sequence into a VAX machine that was shared with the structural biologists. “They didn’t give us any CPU time during the day,” he says. “So you had to queue up your sequence and when they were finished with their work at midnight, the sequence comparisons would run.”

The next morning, Bishop came in and discovered that the gene he’d cloned was a homolog of RecA, the key protein involved in homologous recombination in bacteria. “That blew us all away,” says Kleckner. “Because there were no eukaryotic homologs for RecA ”—despite the fact that many groups had been seeking one.

But Bishop wasn’t the only one who’d snagged a RecA homolog. At Osaka University, Tomoko Ogawa—an old friend of Kleckner’s—and her postdoc Akira Shinohara had also discovered a eukaryotic recombinase. At that point the teams could have opted to compete. But instead, they shared their data—in part, says Shinohara, because Bishop had taken him to a Red Sox game when he’d visited the lab. “We faxed our sequences back and forth,” says Bishop. “And I could tell right away that they weren’t the same.” The genes were clearly related: aligned on the computer they proved to be 45 percent identical. And both are involved in recombination. But the gene discovered by Bishop, called Dmc1, functions only during meiosis, whereas the gene discovered by Shinohara, called Rad51, operates in both meiotic and mitotic cells—results published in back-to-back Cell papers in 1992.

“Everyone congratulated everyone else and gave talks showing American and Japanese flags crossed,” says Kleckner. “It was typical Doug, really. He doesn’t have a personal agenda. He’s just intellectually curious and completely honest in his enthusiasm for science.”

Before leaving Kleckner’s lab for a position at the University of Chicago in 1993, Bishop developed an immunofluorescence technique for visualizing where Dmc1 was located on chromosomes isolated from meiotic cells. “No one had ever done that before: show that you could see individual recombination complexes with antibody staining,” says Bishop.

“There are now huge numbers of papers using this methodology as a tool to study the assembly of DNA repair proteins,” says Kolodner. “And Doug invented it. He has a history of doing interesting stuff—and doing it first.”

The ability to see when and where these proteins bind—and with whom they interact—“helped bridge the gap between the genetics and the biochemistry,” says Stephen Gasior of the University of New Orleans, Bishop’s first graduate student. The technique revealed that Dmc1 interacts with Rad51—and that both require sets of accessory proteins and mediators to help them get to where they need to go. These factors include a set of translocases that use the energy from ATP to truck along the DNA, displacing them from sites that are not in need of recombination. “Dmc1 gloms onto DNA all over the place,” says Terri Holzen of the University of Colorado, Denver, another former student. “So you need to get it off the places where it doesn’t belong.”

Although he’s now adapted the approach to look at recombination on bacterial chromosomes, Bishop isn’t generally wooed by sexy new techniques. “When the first microarray paper came out, we all told Doug, ‘Oh, we should do this,’” says former student Eurie Hong, now head curator of the Saccharomyces Genome Database at Stanford. “But Doug asked, ‘What’s the question you want to answer?’ He felt that you should use a technology to advance your science, not just because it’s cool or trendy. And Doug never compromised on the science.”

And he certainly doesn’t compromise when it comes to publication. “Doug is a very careful guy,” says Holzen. “He likes to make sure that when he submits something for publication, all the I’s are dotted and the T’s are crossed.” When their paper on Tid1—the translocase that clears Dmc1 from DNA to which it is nonspecifically bound—came back with generally positive reviews, Bishop decided that they needed to do a two-hybrid assay to make sure that knocking out the protein’s ATPase activity did not render it unable to interact with Dmc1. “At the time I thought it was irritating,” says Holzen. “And I’m sure I said, ‘This seems really stupid.’ But looking back, it was the right thing to do and I’m very proud of that publication.”

“A lot of these control experiments wouldn’t even end up in the final paper,” says Hong. “They would be ‘data not shown.’ But we could pull it out if a reviewer said, ‘Well, what about this possibility?’” All those careful controls made for strong papers that could take a while to assemble. “Doug’s SPU—the Smallest Publishable Unit—or the minimum amount of work you had to do to write the paper is quite significant,” laughs Hong. “He wants to tell a complete story and he doesn’t want there to be a lot of questions left in terms of the hypothesis you’re trying to prove.”

“Even after you’ve done all the controls, you’d have to make sure that your arguments are crystal clear,” adds former student Sean Sheridan, who now works in the Office of Technology & Intellectual Property at the University of Chicago. “Doug taught me the importance of saying ‘this suggests’ versus ‘this indicates’ because you don’t want to commit to something if you haven’t actually proved it. So it’s important not to overstate what your data show.”

“There are plenty of people willing to publish things that may or may not be perfectly correct,” says collaborator Philip Connell of the University of Chicago. “Doug is the exact opposite of that. He has to make sure that things are exactly right before he publishes. And he questions every single thing he hears. He doesn’t accept something just because someone well known says it.”

Even if that someone is his former mentor. “Doug will often point out to me, ‘No, you can’t say that because of this or that,’” says Kleckner. “Doug has not only made seminal contributions to the field, but he has worked hard to get rid of the junk that people publish that isn’t correct or is misinterpreted.” So in Bishop she can trust. “When Doug tells me something, I believe it,” says Kleckner. “I don’t have to worry about whether he misinterpreted something or didn’t read the literature. If he says so, that’s the way it is.”