Yeast Protein May Be Key to Unlocking Genetic Errors

In the October issue of Nature Structural Biology, research scientists from the Texas A&M University System Health Science Center Institute of Biosciences and Technology and the Howard Hughes Medical Institute and department of biochemistry and molecular biology at Baylor College of Medicine report that the atomic 3-D structure of a protein has the potential to help correct genetic errors that cause human disease.

The protein, known as PI-SceI, is found in the same yeast that is used to make beer and bread, and is a member of a class of enzymes known as homing endonucleases. PI-SceI, like other homing endonucleases, is made by a parasitic DNA element that appears to exist for no other reason but to perpetuate itself. When PI-SceI is made from its gene, it is found in the middle of another yeast protein as an intervening protein or “intein”, and it must first excise itself by “protein splicing” so that its presence does not negatively affect the viability of the yeast. The protein then searches the 13 million base pair yeast genome to find its single DNA recognition sequence, and makes a cut at this site. In doing so, it initiates a “homing” process that leads to the duplication of its own gene and to its eventual propagation throughout the yeast population.

With the sequencing of the human genome, the identification of genetic errors will proceed rapidly. What will be needed next is a means of excising these defective genes in order to replace them with functional copies. Homing endonucleases like PI-SceI may fit the bill since they can act as “molecular scissors” that can locate and cut at a single target sequence among millions of DNA base pairs. When the cell recognizes that a break in the DNA has occurred, it can repair the DNA with a normal copy.

Using X-ray crystallography, Carmen Moure, Ph.D., a post-doctoral fellow in the laboratory of Howard Hughes Medical Institute investigator Florante A. Quiocho, Ph.D., at Baylor, in collaboration with Frederick S. Gimble, Ph.D., in the Center for Genome Research at the IBT, determined the structure of PI-SceI bound to its 36 base-pair DNA recognition sequence. This represents the first structure of an intein bound to its DNA. The structure shows that the protein is divided into two separate domains, where one domain catalyzes the protein splicing process while the other cuts the DNA. The structure suggests that these two domains arose independently but then joined together at some point in evolutionary time to create the intein that is observed today. Furthermore, the structure reveals that both of these domains contact the DNA, which is bent significantly as part of the recognition process.

With the PI-SceI structure in hand, efforts to re-engineer PI-SceI so that it can be regulated inside the cell will be made simpler. At the IBT, Karen Posey, Ph.D., and Gimble have already inserted a molecular switch in PI-SceI that permits its DNA cutting activity to be turned off and on.

“One of our ultimate goals,” Gimble said, “is to use the structural information to create new enzymes derived from PI-SceI that can recognize different target sequences. These molecular reagents would be beneficial to the entire research community for gene targeting, mapping of large genomes and a host of other applications.”

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©2006 Texas Medical Center

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