National Institutes of Health, National Institute of Allergy and Infectious Disease. Ribozyme: Intracellular Catalysis and Antiviral Activity. Given the right molecular methods, ribozymes might become useful antiviral therapeutics. This project will develop new methods for improving the intracellular expression, stability, and effectiveness of hairpin ribozymes that cleave viral RNA. Genetic analysis has confirmed the site of action and mechanism of action of the antiviral ribozymes. These studies are producing methods for expressing and stabilizing catalytic RNA that should help in the development of antiviral therapeutics. (J. Burke, J. Heckman, M. Shields, C. Villarimo, Z. Zhang, J. Han, J. Hathaway)
National Institutes of Health, National Institute of Allergy and Infectious Disease. Hairpin ribozyme: Folding, structure, and mechanism. RNA-mediated catalysis is biologically important but poorly understood. The purpose of this project is to elucidate the structure and reaction mechanism of the hairpin ribozyme, a small catalytic RNA. A molecular model of the reactive core of the ribozyme has been developed and a mechanism deduced and tested. Understanding the molecule will enable researchers to engineer it for special purposes such as cleavage of harmful RNAs in cells. (J. Burke, J. Heckman, K. Hampel, M. Fay, Z. Zhang, D. Lambert, M. Benyah-Essam, P. Chan)
National Institutes of Health, National Institute of General Medical Sciences. Hammerhead ribozyme: Active site assembly and structure. RNA catalysis is biologically important but poorly understood. The purpose of this project is to elucidate the structure of the hammerhead ribozyme active site, and to make progress towards understanding the catalytic mechanism. We have proposed and tested a new model for the chemical mechanism of hammerhead ribozyme catalysis. Functional group substitution, structural assays, and molecular modeling methods are being used to develop and test models for tertiary structure elements, and to define the molecular components of the active site. This project has important potential applications in the development of therapies for genetic and viral diseases. (J. Burke, J. Heckman, K. Hampel, C. Pecore, M. Fay, D. Lambert, J. Han)
Pew Scholars Program. Structural studies of pre-mRNA processing and editing. Pre-mRNAs undergo three post-transcriptional processing reactions before being exported to the cytoplasm: capping, splicing, and polyadenylation. Until now, no structural information existed for any of the polyadenylation factors. The aim of this project is to elucidate the structure of factors of the polyadenylation machinery. We have uncovered two adenine binding sites in mammalian pol(A) polymerase and have proposed a two-state mechanism for nucleotidyl transfer. This structure reveals how this RNA polymerase selects for ATP. (S. Doublie, M. Coseno, J. Meyette)
National Institutes of Health. Crystallographic studies of eukaryotic poly(A) polymerase. In eukaryotes, polyadenylation plays an essential role in the initiation of protein synthesis as well as in the export and stability of mRNAs. Poly(A) polymerase (PAP) is a template-independent RNA polymerase which specifically incorporates ATP at the 3'-end of mRNA. We have solved the crystal structure of bovine PAP bound to an ATP analog at 2.5 A resolution. We have uncovered a two state mechanism for nucleotidyl transfer in mammalian poly(A) polymerase. In addition we have made substantial progress towards expressing large amounts of its processivity factor, poly(A) binding protein. The protein contacts to the nucleotide, together with the metal coordination, provide a structural basis for ATP selection by PAP. (S. Doublie, M. Coseno, J. Meyette)
Human Frontier Science Program Organization. Molecular, structural, and functional analysis of RNP-complexes controlling gene expression. The aim of this project is to combine. biochemistry, X-ray crystallography and cryo-electron microscopy. to decipher the form and function of eukaryotic. splicing factors. (S. Doublie, L. Sevigny)
J. Walter Juckett Foundation. Role of CLF1 in maintenance of genome stability. The information that directs growth and development of all organisms is encoded in DNA. Defects in the duplication of DNA, or in the subsequent distribution of DNA copies to daughter cells, can lead to cancer. The purpose of this project is to understand how a protein known as Clf1p helps to ensure the faithful duplication (a.k.a. replication) of DNA. We have determined that Clf1p helps recruit replication factors to origins of DNA replication, thereby ensuring that replication begins at the appropriate stage in the cell cycle. This work will help scientists understand how defects in cell cycle control and DNA replication can lead to cancer. (D. Pederson, W. Zhu, L. Bardot)
National Institutes of Health, National Cancer Institute. Processing of damage by translesion DNA synthesis. Bypass of a DNA lesion by DNA polymerases is essential to express a mutation and to initiate carcinogenesis. The aim of this project is to elucidate how oxidative DNA lesions interact with DNA polymerases using x-ray crystallographic approaches. Results show that the most common cellular DNA damage, a site of base loss, blocks a DNA polymerase by failure to translocate out of the active site. Insights into these fundamental interactions provide clues to the roles of DNA damage in cancer and aging. (S. Wallace, S. Doublié, J. Bond, V. Bandaru)
National Institutes of Health, National Cancer Institute. Repair of DNA damage induced by ionizing radiation. Whether a cell dies from DNA damage or survives to become a mutated or cancer cell depends upon the efficiency of repair of the damage. This project is studying the properties of repair enzymes of the Nth Superfamily and the damages they remove, and determining how these damages cause cellular mutations. Researchers have defined enzymatic repair pathways for oxidized cytosines, delineated properties of a newly discovered human repair enzymes and determined the crystal structure of one of these, and examined the consequences of repair of closely opposed damages. These studies should provide insights into the molecular mechanisms underpinning cancer and other human diseases. (S. Wallace, J. Blaisdell, S. Kathe, V. Bandaru, J. Bond)
U.S. Department of Energy. Damage recognition, protein signaling, and fidelity in base excision repair. Large numbers of oxidative DNA damages occur during normal metabolism and the damages produced initiate the carcinogenic process. The purpose of the studies is to understand at the atomic level the structures and mechanism of action of the proteins involved in the repair system that removes oxidative damages from DNA. The results will provide fundamental insights into how these DNA repair enzymes recognize and. repair a broad array of oxidative DNA lesions. (S. Wallace, M. Kennedy, S. Ni)
National Institutes of Health/National Cancer Institute. Structure, Function, and Evolution of DNA Repair Enzymes. DNA repair processes are highly conserved across all species and whether or not a DNA damage is repaired or not determines whether or not a cell goes on to die, be mutated, or become a cancer cell. This series of projects are using bioinformatic, x-ray crystallographic and enzymatic techniques to steady the properties of several important classes of the DNA repair enzymes including DNA glycosylases and repair recombinases. Several new human enzymes have already been identified and the crystal structure of one of these determined. These studies should provide insight into fundamental processes underpinning cancer and other human diseases at the atomic level. (S. Wallace, S. Doublié, S.W. Morrical, J. Bond, M. Rould, R. Kocherlakota, S. Robey-Bond, J. Liu, S. Feehan, R. Barrantes-Reynolds, J. Blaisdell, W. Cooper, J. Meyette, P. Laverty, F. Roy, D. Stern)
11 projects