Massa Shoura was lead author of the paper published in the journal Nucleic Acids Research.

In
a new study, UT Dallas researchers outline how they used fluorescent
molecules to “tag” DNA and monitor a process called DNA looping, a
natural biological mechanism involved in rearranging genetic material in
some types of cells.

The
UT Dallas “tag and track” method 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.

Until
now, scientists primarily had “snapshots” of the initial and final
stages of DNA loop formation, with only limited information about what
happens during the intermediate steps, said Dr. Stephen Levene,
professor of bioengineering, molecular and cell biology, and phyiscs at
UT Dallas. He is senior author of the study, published online and in an
upcoming issue of the journal Nucleic Acids Research.

“Scientists
have known for more than 30 years that DNA looping is an important part
of molecular biology and gene regulation, but until our work, there
have been few serious attempts to understand the basic biophysics of the
process,” Levene said.

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, Levene said.

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 is so good at forming DNA loops and
excising genetic material that scientists routinely use it 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.

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,” Shoura said. “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,” Levene said.

Other
UT Dallas researchers from the Department of Molecular and Cell Biology
who participated in the study were senior scientist Dr. Alexandre
Vetcher; doctoral students Stefan Giovan, Farah Bardai and Anusha
Bharadwaj; and former undergraduate student Matthew Kesinger. The
National Institutes of Health and the National Science Foundation funded
the research.

 Measurements of DNA-loop formation via Cre-mediated recombination

Source: University of Texas Dallas