A team of researchers in VCC's Genome Stability & Expression Program was recently awarded $7.5 million in the form of a Program Project grant. The grant, awarded to the researchers by the National Cancer Institute (NCI) over a five-year span, will fund a study using biochemical, computational, and structural biology methodologies to determine how three families of DNA enzymes repair damage caused by ionizing radiation.
The award—one of just several Program Projects currently funded at UVM—was made to Susan Wallace, PhD, program leader of the Vermont Cancer Center (VCC) Genome Stability & Expression Research Program, and four other VCC members within the Program: Jeff Bond, PhD, research associate professor of microbiology and molecular genetics; Sylvie Doublié, PhD, associate professor of microbiology and molecular genetics; Scott Morrical, PhD, professor of biochemistry; and Mark Rould, PhD, research assistant professor of molecular physiology and biophysics.
There are two types of repair systems that fix the DNA damage caused by radiation: base-excision repair, which removes damaged bases, and repair by homologous recombination, which repairs double strand breaks (the defects in individuals carrying BRCA1 and BRCA2 gene mutations associated with hereditary breast cancers). The Program Project, which consists of three projects and three cores, will examine both types. Wallace, principal investigator for the grant, is the leader for Project 1, "Specificity of the BER Oxidative DNA Glycosylases," and Doublié is the leader for Project 2, "Structural Basis for the Substrate Specificity of the BER Enzymes;" both of which will study base-excision repair processes. The second type of DNA repair is to be studied in Project 3, entitled "Structure and Function of Homologous Recombination Enzymes," with Morrical as the leader and Rould serving as senior investigator. A Bioinformatics Core led by Bond; an Expression, Characterization, and Crystallization Core led by Doublié; and an Administrative Core led by Wallace support all three projects.
The researchers will study the HhH-GPD Nth superfamily of DNA glycosylases, the Fpg/Nei family of DNA lycoslyases, and the RecA family of recombinases. These particular enzymes were chosen for study due to their high degree of conservation across the animal kingdom. "We're using this fact—that these proteins are highly conserved across phyla—to help us understand how the proteins work," says Wallace. "The DNA glycosylases, for example, are able to see a single damaged base in a sea of normal nucleotides. So we're trying to determine how these proteins recognize and know to bind specifically to their damaged DNA substrates.”
Project 1 is primarily a biochemically oriented project; proteins will be cloned, expressed, purified, and characterized. The effort will be based on guidance from computational analysis in conjunction with phylogenetics, provided by the Bioinformatics Core, as well as on high throughput assays provided by the Expression Core.
With Project 2, the scientists will use x-ray crystallography to attempt to determine the three-dimensional structures of particular proteins that showed promise in Project 1. Those that can be crystallized will be analyzed for their unique DNA repair specificities.
Project 3 aims to look at recombination proteins using similar biochemical, computational, and structural approaches. "A key goal of this project is to capture an atomic-resolution image of a RecA-DNA complex," says Rould. "That's the Holy Grail of the recombination field." Another goal of the project is to understand differences in the mechanisms of related recombinase enzymes. According to Morrical, "The human genome encodes six different versions of the main Rad51 recombinase and mutations in any of them cause a DNA repair defect."
According to Wallace, the grant received by these VCC researchers is one of just three program projects funded by the NCI that is highly dependent on structural biology. The others are at Stanford University and the University of California, Berkeley. Wallace says that the NCI was especially excited about this program project's unique use of computational and phylogenetic approaches to look at DNA repair.
"I believe ours is the only biochemistry-crystallography project in the country that is based on a computational approach," Wallace says. "That's the aspect of it the reviewers praised the most. We're ahead of the curve from that perspective and we have Jeff Bond to thank for it. He is a computational biologist with a degree in biochemistry so he truly understands what the rest of us are trying to do. These days, you have to rely on people with specialized talent to push the envelope in our field."
Understanding the repair of radiation-induced damages is important to an improved understanding of cancer for two reasons: radiation is an accepted treatment modality for some cancers and the ability of tumors to repair the radiation-induced damage has a direct bearing on patient outcome. Secondly, our society's exposure to low levels of radiation is increasing due to exposure to the sun, radon, some medical diagnostic procedures (e.g., stress tests, thyroid tests), and nuclear waste. Since radiation is a known carcinogen, it is essential for us to learn how cellular repair systems cope with radiation damage to humans as a result of medical and environmental exposures.
"If we knew how to up the efficacy of some of these repair processes, which is what people try to do when they take anti-oxidants, we might be able to prevent some cancers," says Wallace. "Also, if we could down-regulate the repair process to make it less effective, we'd make radiation therapy for cancer patients more effective. You see, when an individual has a tumor and is being treated with radiation therapy, doctors don't want repair to be working! So our research, though very much in the basic realm, can be helpful in two very different ways related to cancer."
Rould says that Project 3's recombination studies have the potential to inform cancer understanding in additional ways. Besides repairing genetic damage, genetic recombination is the primary means by which bacterial and cancer cells acquire resistance to treatment such as chemotherapy. When cancer cells are challenged with a new drug, they respond by increasing the rate with which they mix-and-match portions of their genes, in an effort to produce new enzymes that will counter the chemical assault. This genetic swapping is itself carried out by enzymes called recombinases—truly the engines of change and evolution at the molecular level.
"One of the goals of this Program Project is to understand—actually, to see—how the recombination machinery exchanges DNA between similar, but not identical strands," says Rould. "Once we know how this works, we'll be better poised to control it, perhaps leading to the development of a new class of pharmaceuticals that will prevent tumors from 'escaping' current treatments."