Fluorescent light 'tags and tracks’ DNA looping

Aug. 13, 2012
University of Texas at Dallas (UT Dallas) researchers used fluorescent molecules to “tag” DNA and monitor a process called DNA looping. The work not only sheds light on how DNA loops form, but also might be adapted to screen drugs for effectiveness against certain viruses that shuffle genetic material, such as HIV.

University of Texas at Dallas (UT Dallas) researchers used fluorescent molecules to “tag” DNA and monitor a process called DNA looping. The work not only sheds light on how DNA loops form, but also might be adapted to screen drugs for effectiveness against certain viruses that shuffle genetic material, such as HIV.

DNA looping is a mechanism common in many instances of natural gene-splicing. Proteins within cells—or proteins made by invading viruses—latch onto specific docking points on a DNA molecule. They bring those points together to form a loop, and then snip out the genetic material between the points while reconnecting the now-loose ends.

DNA loop formation is especially important in organisms whose genetic material is circular, including some bacteria and viruses. Human DNA is linear, but the possibility that DNA looping takes place in human cells is an ongoing area of investigation, says Stephen Levene, Ph.D., professor of bioengineering, molecular and cell biology, and physics at UT Dallas.

Levene and UT Dallas doctoral student Massa Shoura, the lead author of the paper, used a protein called Cre in their experiments. Cre is made by a virus that infects bacteria and does well at forming DNA loops and excising genetic material that it is routinely used to delete genes from laboratory animals, which are then used to study the role of genes in human disease.

Levene and Shoura engineered isolated segments of DNA to contain Cre’s docking points. They also inserted into those points a molecule that fluoresces when exposed to certain wavelengths of light. By monitoring the changes in fluorescence, the researchers could watch the steps of the loop formation.

Stephen Levene, Ph.D., and doctoral student Massa Shoura have devised a way to track the formation of DNA loops. (Image courtesy of UT Dallas)

The information the researchers have gleaned is not only useful for understanding basic biology and genetics, but also might lead to more efficient methods for screening potential new drugs for anti-HIV activity.

Once inside a host cell, HIV produces an enzyme similar to Cre, called an integrase. As its name suggests, the integrase slices into the host’s DNA and inserts HIV’s genetic material.

“Our fluorescent-tag technique could be used in the lab to more closely examine how HIV inserts itself into the host’s genome,” says Shoura. “By labeling and monitoring the process, we also could test drugs designed to interfere with the integrase.”

“We estimate that using fluorescence-based methods such as this for drug screening could be as much as 10,000 times more efficient than methods that are currently used,” says Levene.

The work has been published in the journal Nucleic Acids Research; for more information, please visit http://nar.oxfordjournals.org/content/early/2012/05/24/nar.gks430.abstract?sid=169fc5a9-ddfc-4127-89e4-76f56b7e67a5.

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