With colorful spirals, twists and segments, an x-ray crystallography image looks more like a brain teaser puzzle than a powerful scientific tool. The comparison is appropriate; x-ray crystallography structures – atomic-level visuals of proteins and DNA – need to be solved and in that regard, the University of Vermont’s Sylvie Doublié, Ph.D., is a puzzle master. During her career, she has helped solve an estimated 80 structures, several of which have resulted in critical discoveries related to the molecular activities that lead to DNA damage, mutations, and often cancer.
At a reception held in May, Doublié, a professor of microbiology and molecular genetics, was honored as one of four 2014-2015 University Scholars at UVM. She is excited to be receiving this honor during a milestone year: the 100th anniversary of x-ray crystallography.
X-ray crystallography allows molecular biologists to zoom in on a protein, gaining both a general representation of the overall shape and form of a protein as well as a detailed view at the atomic level. To get there, a scientist must first mass produce the protein of interest, then grow crystals, put the crystalline sample in the x-ray beam, and using companion x-ray crystallography computer software, generate an atomic model of the protein with x,y,z coordinates for each of the thousands of atoms in the molecule.
When x-ray crystallography was first suggested to her as an undergraduate thesis topic at the University of Paris in France, Doublié admits she wasn’t interested. “In my mind, crystallography meant analyzing the rocks you see in geology museums, but I didn’t know the extent of what you could do with the technology,” she says. Then she studied the process, and quickly learned she could look at virtually any protein at the atomic level with the technology. From that point forward, she was hooked.
Doublié specializes in DNA damage, repair and replication. A University Scholar nomination letter shared that her colleagues refer to her as “a leading crystallographer in her field and one of the premier structural biologists in the DNA replication and repair field.”
She and her team are interested in how DNA polymerases – enzymes or proteins that copy DNA in a genome – interact with DNA and what happens when DNA is damaged. This damage comes from daily living – breathing in air, being exposed to the sun – so DNA is constantly under attack. With x-ray crystallography, explains Doublié, “you can zero in on that damage and look at how the protein interacts with it; the structure can illustrate what really happens at the atomic level.”
Among the many solved structures in Doublié’s repertoire is one, published in the Journal of Biological Chemistry in 2012, which involves the repair systems that scan for damage in DNA. While scientists are not sure exactly how these systems perform this task, their findings provided Doublié and her colleagues with a better understanding of the system’s processes. According to Doublié, when DNA damage occurs, enzymes called glycosylases will flip out – or evert – and repair the damaged site. She and her team wondered how the system was able to locate the damage and not take out normal bases in the double helix of the DNA. From an analysis of the crystal structure, they determined that the enzyme senses the problem while the damaged base is still inside the double helix. Once the damaged area is flipped out and bound by the enzyme, only damaged bases are taken out, thereby preserving the normal bases.
Another project, which resulted in a 2010 publication in the Proceedings of the National Academy of Science (PNAS), unlocked a mystery regarding the direction of a specific polymerase. While all DNA and RNA enzymes copy DNA or RNA in one direction – from 5 prime to 3 prime – UVM’s Nicholas Heintz, Ph.D., professor of pathology, studied a protein called THG1 that goes the other way – from 3 prime to 5 prime. Doublié collaborated with him and Jane Jackman at Ohio State University to work on solving the structure so that she could see what the reverse polymerase looks like. While she and her colleagues expected there would be a very different fold in the structure for this protein, they were surprised to discover that it looks very similar. While the overall shape of THG1 and that of a canonical polymerase differed, the active site or “business end” – the part responsible for the reaction – was the same.
“Nature took the same fold – recycled it – and did something different with it,” remarks Doublié. “For me, as a polymerase ‘geek,’ it makes sense, because it’s a good fold.”
Solving structures and viewing folds has become a lot easier for Doublié and her colleagues since November 2013. Thanks to a very generous gift from the Trunk Foundation, they were able to purchase a new x-ray crystallography system after working with nearly-obsolete, 16-year-old technology.
Among her other frequent collaborators are Department of Microbiology and Molecular Genetics members Susan Wallace, Ph.D., chair, David Pederson, Ph.D., Scott Morrical, Ph.D., Jeffrey Bond, Ph.D., Joann Sweasy, Ph.D, and Greg Gilmartin, Ph.D.
Though conducting most of her work in the small state of Vermont, Doublié has – not surprisingly – earned an international reputation. A comment in another nomination letter states that she “is particularly known for her accomplishments in the area of enzymes that operate on DNA and RNA to carry out DNA replication, DNA repair, and RNA processing” and “her work has led to numerous fundamental discoveries concerning the structure and mechanism of DNA polymerases.”
Doublié, who also serves as co-director of the undergraduate biochemistry program in the College of Agriculture and Life Sciences, joined the UVM faculty in 1998. After earning an undergraduate degree from the University of Paris, where she worked with Anita Lewit-Bentley, Ph.D., she received a Ph.D. in biochemistry and biophysics under the mentorship of Charles Carter, Jr., Ph.D., at the University of North Carolina School of Medicine. She then completed postdoctoral work with Stephen Cusack, Ph.D., at the European Molecular Biology Laboratory outstation in Grenoble, France, and at Harvard Medical School with Thomas Ellenberger, D.V.M., Ph.D., where she solved the crystal structure of the ternary complex of T7 DNA polymerase, a study published in 1998 in Nature. In 2000, Doublié received a prestigious Pew Scholar in the Biomedical Sciences award. Last year, she was elected a member of the Vermont Academy of Science and Engineering. She is the author of more than 65 journal publications.
Of her journey to this point, Doublié shares that “science is not a straight road – a lot happens in the hallways and at meetings and you take a different path.”
“When I look at the work, I think of the people – the students, technicians, and fellows – and how each one of them taught me something along the way,” says Doublié, who has mentored nine undergraduate students, eight graduate students, seven postdoctoral fellows and 11 technicians since joining UVM in 1998.
The University Scholars program recognizes distinguished UVM faculty members for sustained excellence in research, creative and scholarly activities. Scholars are selected by a faculty panel based upon nominations submitted by UVM colleagues.
