Scientists at the University of Pennsylvania Medical Center have determined the molecular structure of an important enzyme used in genetically engineering animal models for studies of human diseases. Knowing the three-dimensional arrangement of the protein--Cre recombinase-- will one day aid in designing better research models. The research team--led by Gregory D. Van Duyne, PhD, Assistant Professor of Biochemistry and Biophysics--has reported its findings in the Sept. 4, 1997 issue of the journal Nature.
Cre recombinase is a member of a large family of proteins used by bacteriophages, viruses that infect bacteria. Phages use these enzymes to splice their own genes in and out of the host bacterial genome and to maintain their own genetic integrity during bacterial replication. "What was really lacking was a structural model of this enzyme to tie in with the biochemical and molecular genetic data that has been amassing over the last several years," says Van Duyne. "What everyone's been waiting for is to see what one of these recombinases looks like bound to DNA. We have it not only bound to its DNA substrate, but we've trapped it after it has completed one step of the recombination reaction."
Freeze-Framing Reaction Led To Development of 3-D Structure
Using a molecular trick to stop the recombination reaction--the process of cutting and splicing together genetic material from different sources--the researchers were able to freeze the process at an early step. Getting such a complete three-dimensional picture of even one step in this complicated reaction has never been accomplished until now.
Because of the simplicity of Cre recombinase's splicing mechanism--it works alone, requiring no additional enzymes--the protein has been used extensively in genetic engineering in recent years. "It's now possible using Cre recombinase to produce a transgenic mouse where you target, for example, a suspected cancer-causing gene and ask the question: What happens if I turn this gene off or on at a specific time during development just by controlling whether Cre recombinase gets expressed?," remarks Van Duyne. "Cre recombinase has made this type of experiment possible, and now it's being done almost routinely." The hope is that by understanding how Cre identifies the specific site on the DNA that it's supposed to cut, molecular biologists will be able to improve genetic control of transgenic animal models.
Recombination: Less Complicated Than Originally Thought?
In addition to the practical advances this detailed structural picture will provide, the discovery lends strong support to a recently proposed new way of looking at how recombination occurs. Where the DNA strands cross during the recombination reaction is called a Holliday junction. For many years, people thought that the junction would be formed at one end of the DNA strand and that that junction would slide along to the other end before the next step in the reaction could continue. "However, our structural data suggests that the junction stays put," explains Van Duyne. The structure that the Van Duyne team captured is one step before the formation of the Holliday junction.
The structure of the complex also alludes to a recombination reaction that is much more simple than previously thought. "The really unexpected part of the structure was--and this is still speculative--that the enzyme accomplishes its task of rearranging the DNA substrate with only a few uncomplicated contortions," says Van Duyne. The thinking had been that the recombinase-DNA complex has to go through a series of complex positional changes between each step of the reaction.
"Now that we have a three-dimensional model, the next step is to reinterpret the biochemical data and see if there is a different way of explaining the recombination reaction in terms of a simpler protein-DNA architecture," says Van Duyne. In the future, the Van Duyne team plans to elucidate the structure of later steps in the recombination reaction.
This work was conducted in the Johnson Research Foundation, a funding and research organization within Penn's Department of Biochemistry and Biophysics that concentrates on the study of physics as it applies to medicine.
Dr. Van Duyne can be reached at 215-898-3058
An electronic image of the Cre recombinase-DNA complex structure will be available on the web after September 4 at: http://www.med.upenn.edu/biocbiop
The University of Pennsylvania Medical Center's sponsored research ranks fifth in the United States, based on grant support from the National Institutes of Health, the primary funder of biomedical research in the nation. In federal fiscal year 1996, the medical center received $149 million. In addition, for the second consecutive year, the institution posted the highest growth rate in research activity--9.1 percent--of the top-ten U.S. academic medical centers during the same period. News releases from the medical center are available to reporters by direct E-mail, fax, or U.S. mail, upon request. They are also posted to the center's home page (http://www.med.upenn.edu) and EurekAlert! (http://www.eurekalert.org), an Internet resource sponsored by the American Association for the Advancement of Science.
Journal
Nature