Recorded: 01 May 2000
Jeff: The outgrowth of those times in Cold Spring Harbor that I’m continuing on now is that the switching process is initiated by a double strand break in cerevisiae. You’ll hear a slightly different story from Amar. Those observations we made in 1981, or about that time while at Cold Spring Harbor. That led to a whole field, probably dozens of laboratories who used the HO gene that Amar referred to. It turned out to be a double strand endonuclease. A lot of people used that as a site specific endonuclease because it has a very large recognition site so that it only cuts once in the yeast genome. You can put it into mice or put it into Drosophila, and it will find its recognition site and cut it. And I followed that process into the study of recombination initiated by double-stranded breaks.
So my laboratory here studies mitotic recombinant in DNA damage repair processes. About three or four years ago, we demonstrated—we asked the question, “What DNA polymerases are involved in the recombination process?” In almost every model as drawn on the chalkboard by people who that love this kind of topic, a portion of the model is dotted lines and the dotted lines are the new DNA synthesis that is associated with this process. We asked the question whether the new DNA synthesis associated with recombinational repair has as high a fidelity as the DNA synthesis associated with S-phase replication. The answer was no, not at all. It’s a hundred-fold more likely to make an error this S-phase DNA replication.
That led us into the topic that is one of the current areas of research in the lab is: What are the polymerases involved in [synthesis]? What factors are associated with them and what factors [are] missing from them that makes this a more error-prone process? That constitutes about a third of what is going on at the lab and it’s a direct outgrowth of this observation in cerevisiae that homothallic switching is initiated by a double strand break, the preciseness of a double strand break. That’s the tool that allows us to make those breaks and then study how they are resolved.
Amar: So, in the same way, we continued with the mating type switching. So I got interested in wanting to know if these things which we were studying whether they’re useful elsewhere in some other system. Wonderful genetics in deficient yeast Schizosaccharomyces pombe existed and brought David Beach coming to my lab. We did some things with mating type. Beach went to the cell cycle study, so I brought that with me here.
This organism [S. pombe] is also wonderful in terms that all the tools of molecular biology and genetics which are applicable in yeast and cerevisiae are equally well applicable, but the pattern of switching and the arrangement of the cassettes is very different in this organism, compared to cerevisiae. So at the very least, we figured that we could discover some new things and how [a] similar system can evolve. That guess turned out to be correct that the mechanism of switching is here, in this case, is by something happening at the MAT lobe, which makes it cleavable, which initiates mating type switching and recombination again.
Secondly, we found out that silencing is much more extensive in this organism which is now, much more, compared to cerevisiae. We identified many of the concepts and factors which are involved in silencing which are probably all these things—whatever we talked about, even though they looked esoteric. They’re all useful in biology and in other systems… That’s why we humans have different tissues that turn on different genes and turn off other genes. And probably that is accountable to silencing and also this is one of the wonderful things which we found in fission yeast (this is going back to Jim Watson): When cells divide, the two sisters are different from each other. Simply because one sister inherits the original Watson strand and the other sister inherits the original Crick strand at the mating type locus and that’s why they are different. We found that people in the lab decided that it was site-specific alkaline biomodification or a neck in DNA. Jacob Dalgaard had been working on it and a lot of other things. Here two sisters are dividing simply because of Watson and Crick DNA strands are complementary and not identical. And that makes the two sides different.
Going from there as a side project, I got interested in ideas about why some people are left-handed. It has to do with two hemispheres of the brain. We think that it’s the same question, if you could figure out how the two sisters were different, conceptually it’s the same question how two sides of the body are different. We have a heart on this side and not that side. We’ve been doing more than theoretical work on it, but I think it will be interesting if we can push this principle which we are learning from yeast into a much more complicated system.
Otherwise nobody else would do it because you have to be trained in particular setup principles. Only then you can address that, because not everything can be done. Just to clone a gene, you have to know what to clone first. We try to do it with those kind of things. What is the area to be addressed molecularly? There are still a lot of things which need to be done before you know what to clone. Cloning—for me, that’s a set project; that’s straight forward. But before that, you have to know what to clone and how to clone it; it’s also important. So yeast training allows you to do that.
Jeff: We had a saying while we were at Cold Spring Harbor, because there were a lot of areas of yeast as a research organism that blossomed and many topics blossomed during that period. We always had the feeling that we didn’t have to leave yeast, leave mating type to study any number of things because it would turn out that all of those things end up some part in the yeast system and the mating type system. It’s been true many of the issues of cell biology and chromatin organization and DNA damage repair and recombination and site specific termination and DNA binding protein, positive and negative regulators—they’re all mating type. They’re all in the mating type story. You had access to any of those intellectual entertainments still within the realm of saying, “I work on mating type cloning control.” You didn’t have to take up a new title, of “I work on chromatin.” It was all part of this story.
Amar Klar and Jeff Strathern worked together in the Cold Spring Harbor Yeast group from 1977 till 1984 where they made outstanding discoveries about the mechanism of mating type switching in yeast.
Amar Klar, is a leading yeast geneticist, concerned with the molecular biology of gene silencing and mating-type switching. Klar came from India to the University of Wisconsin in 1975 to receive his Ph.D. in bacteriology. From 1977 to 1984, he worked with Jeff Strathern and Jim Hicks in the Cold Spring Harbor Yeast Group studying the mechanism of mating type switching. Klar served as Director of the Delbruck laboratory from 1985 to 1988.
He left Cold Spring Harbor to join the ABL-Basic Research Program as Head of the Developmental Genetics Section. In 1999, Klar joined the National Cancer Institute Center for Cancer Research and is now a Principal Investigator in the Gene Regulation and Chromosome Biology Laboratory at NCI-CCR.
Jeffrey Strathern, a leading yeast geneticist, obtained his Ph.D. from the Molecular Biology Institute at the University of Oregon in 1977 and then moved to Cold Spring Harbor Laboratory, where he became a Senior Staff Member with the yeast genetics laboratory.
In 1984, he joined the ABL-Basic Research Program at the NCI-FCRDC. His research remains centered on aspects of gene regulation and genetic recombination as revealed by studies in yeast. In 1999, Strathern joined the Division of Basic Sciences, NCI. Strathern worked together with Amar Klar and Jim Hicks in the Cold Spring Harbor Yeast group from 1977 to 1984 where they made outstanding discoveries about the mechanism of mating type switching in yeast.