A “tag and track” method using fluorescent molecules 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. Dr. Stephen Levene, a bioengineering, molecular and cell biology and physics professor at the University of Texas at Dallas, and doctoral student Massa Shoura developed the method to monitor the stages of DNA looping, a natural biological mechanism common in many instances of gene-splicing. Dr. Stephen Levene and doctoral student Massa Shoura have devised a way to track the formation of DNA loops, a natural biological mechanism involved in rearranging genetic material in some types of cells. (Images: UT Dallas) In the formation, 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 by snipping out the genetic material between the points and reconnecting the now-loose ends. Until now, scientists had snapshots of only the initial and final phases of the formation, with only limited information about what happens during the intermediate steps. “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. The loop formation is especially important in organisms whose genetic material is circular, including some viruses and bacteria. Although human DNA is linear, the possibility that DNA looping takes place in human cells is under ongoing investigation. The scientists used a protein called Cre, made by a virus that infects bacteria. Cre is so good at forming DNA loops and excising genetic material that scientists use it routinely 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 inserted a molecule into the points that will fluoresce when exposed to certain wavelengths of light. By monitoring the changing fluorescence, they observed the steps of the loop formation. Massa Shoura is the lead author of the Nucleic Acids Research paper that describes a “tag and track” method that sheds light on how DNA loops form, which could lead to more efficient methods for screening potential new drugs for anti-HIV activity. Not only do their observations shed light on basic biology and genetic functions, but their findings could also lead to more efficient methods of HIV drug screening. A fluorescent tag technique based on this study could be as much as 10,000 times more efficient than conventional drug screening methods, Levene said. HIV, once it is inside a host cell, produces an enzyme similar to Cre that is called an integrase. This enzyme 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.” The study was funded by the National Institutes of Health and the National Science Foundation. It appeared online in Nucleic Acids Research. For more information, visit: www.utdallas.edu