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A newly described method linearizes circular chromosomes and caps them with telomeres

Reporter: Aviva Lev-Ari, PhD, RN

Synthetic biologists often work with circular chromosomes to engineer genetic material because they’re stable and easy to manipulate, but they don’t resemble the natural shape of chromosomes in eukaryotes. Reporting in PNAS this week (November 5), Jef Boeke of NYU Langone Medical Center and postdoc Leslie Mitchell designed a tool, which they dubbed the telomerator, that straightens circular yeast chromosomes and adds telomeres to either end.

“To convert circular DNA to something more akin to a natural chromosome is appealing,” said Timothy Lu, a synthetic biologist at MIT who was not involved in the study. Lu said the telomerator could help advance a number of goals, from designing artificial chromosomes that encode complex pathways to testing the significance of telomere location in the genome. “It’s really a platform technology for downstream applications.”

The telomerator includes an endonuclease target—the site where the DNA loop will be severed—flanked by telomere seed sequences that form the basis of telomere construction. The telomerator is inserted into a gene of interest in the circular chromosomes, and when an endonuclease cuts the sequence at the recognition site, each exposed end carries a seed sequence on which to build telomeres. “The reason you can actually linearize the molecule and produce a stable molecule is because this telomere seed sequence gets exposed,” explained Mitchell. She and Boeke engineered the telomerator so that it would be induced only in the presence of galactose, giving scientists an easy way to turn it on by simply changing the growth medium.

The researchers tested the telomerator on a synthetic chromosome designed a few years earlier, cutting it at 54 different genes. For 51 of the permutations, “we got pretty happy yeast at the end of the experiment,” said Boeke. For another three, it appeared that the proximity of essential genes to the new telomere interfered with their expression, a phenomenon known as telomeric silencing. Blocking telomeric silencing rescued the cells’ growth. “I would say we’ve worked out most of the kinks for yeast,” said Boeke. “All you need to decide right now is where you want to put it.”

Yo Suzuki, a synthetic biologist at the J. Craig Venter Institute, said that linearizing DNA is important to avoiding problems in meiotic recombination that can emerge with circular chromosomes. In particular, crossover events between two circular chromosomes can result in a chromosome with two centromeres, which would break during chromosome segregation. “If you have a linear molecule in yeast, you don’t have that problem,” Suzuki said.

Source: www.the-scientist.com

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