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NYU Langone Researchers Build Tool to Linearize Circular DNA Molecules for Synbio Applications

NEW YORK (GenomeWeb) – Researchers from New York University Langone School of Medicine have developed a synthetic biology method to linearize circular DNA molecules in vivo.

The new tool, dubbed the telomerator, "inducibly and precisely linearizes circular DNA molecules in vivoyielding derivatives that encode functional telomeres at each end," they explained in a study published online last week in Proceedings of the National Academy of Sciences. Further, the telomerator offers a new method for building synthetic chromosomes, which typically are constructed as circular molecules, "as telomerator-mediated linearization permits specific positioning of genes in order to modulate their expression."

In a statement, NYU Langone said that the telomerator could also improve the study of yeast genetics and aid scientists researching how genes and chromosome interact with each other.

Jef Boeke, director of the Institute for Systems Genetics at NYU-Langone, who led the research, told GenomeWeb Daily News that as far as he knew, there is no other method for linearizing a circular molecule in vivo. While the use of circular molecules have been very successful in biotechnology and molecular biology, "pretty much all of the cloning that's ever been done has been done on circular molecules," he said.

Engineering yeast to create a complex molecule, for example, can be extremely complicated. The telomerator, Boeke said, potentially could significantly simplify that process.

Currently, functional and stable yeast chromosomes have been synthesized in a circular format with the telomeres cut off so that they can be reproduced uniformly for easy experimentation within bacteria, whose chromosomes are circular. "What [the telomerator] allows you to do is, at the end, to turn that into a linear molecule where it's going to be totally stable," he said.

In addition to complicated pathways, using circular molecules, especially in eukaryotes, have other drawbacks. When yeast cells go through meiosis, the chromosomes will not segregate properly if the molecule is in a circular shape because they will recombine with each other forming dicentric chromosomes that are unstable, Boeke said.

"That's one reason eukaryotic cells have linear chromosomes," he said, and by changing circular molecules into linear ones, they will more closely resemble the structure of more complex organisms including humans.

For their research, he and his colleague Leslie Mitchell chose to work with yeast to develop the telomerator because of its status as a model microorganism for human genetics and because of the ease with which it can be manipulated. The telomerator is a device comprising telomere seed sequences and the site that's cut by an enzyme, which can be plugged into a circular chromosome. It is made up of approximately 1,500 chemical base pairs linked together that can be inserted at any position on a circular DNA and almost anywhere among a chromosome's other genes.

The telomere seed sequences are a critical component of the telomerator as they are exposed when the telomerator is activated, and by flipping a chemical switch, a circular DNA can be converted into a "linear form of defined structure, which has two natural looking telomeres," Boeke said.

The researchers tested the telomerator on a circular synthetic yeast chromosome of about 90,000 base pairs to determine whether the tool could cut the chromosome and straighten it at 54 different locations. "And we were essentially able to convert every single one of those circles into a linear [shape] of slightly different structures," Boeke said.

When the researchers began the project, they had two primary objectives, he said. One was to answer whether it could even be done. Second, they sought to discover whether any prohibitions existed to where a circular molecule could be linearized and functional chromosomes could be obtained.

What they found was that some of the cells with the linearized chromosomes had growth defects, a result that earlier work by others had suggested would happen. Putting telomeres next to genes can weaken the heterochromatin, "which is deleterious for gene expression," Boeke said. Some of the genes on the chromosomes are essential genes, which when located near the end of a linearized molecule led to the growth inhibition. By knocking out the genes required for the formation of the heterochromatin, though, the researchers discovered that "all of these strains grew just fine."

For now, Boeke said the telomerator is a tool for building synthetic chromosomes using yeast. While in principle "there's no reason you couldn't do this in a mammalian cell," he acknowledged that there are challenges "having to do with the repetitive structures of telomeres … [and] animal and plant systems tend to be more difficult to manipulate than yeast."

Still, the telomerator could potentially offer a tool for researchers to open a circular molecule in "as many different places as the investigator would like … and in principle, this approach could be applied to any eukaryotic cell to make very large linear chromosomes," Boeke said. "We're looking ahead to much higher and larger level genome engineering over the next 10 years … and we think this is going to be a critical tool for doing that."