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Molecular Mechanisms in DNA and RNA Processing
Summary: Gregory Van Duyne is interested in the molecular mechanisms that cells use to maintain and process the information contained in their chromosomes.
We are interested in understanding on a structural and biochemical level how chromosomes are maintained by living cells and how the RNA molecules transcribed from protein-coding genes are processed and delivered to their appropriate destinations. Maintenance of a cell's genetic material involves a number of highly regulated processes, including replication and segregation of the chromosomes during cell division, repair of broken or defective DNA strands, condensation and expansion of chromosomes during the cell cycle, and rearrangements of the chromosomes as part of programmed cellular specialization. After protein-coding genes on the chromosomes are transcribed to form messenger RNA (mRNA) in eukaryotic cells, noncoding intron sequences need to be removed, the spliced RNA products are exported from the nucleus to the cytoplasm where they can be translated to proteins, and in some cases the mRNA molecules are localized to specific regions of the cell where they determine the developmental fate of a progeny cell. We are attempting to understand the underlying molecular mechanisms cells use to carry out these processes. We use x-ray diffraction methods to establish three-dimensional models of macromolecular assemblies relating to biological questions and then develop mechanistic and functional models that can be tested.
Site-Specific Recombination
Site-specific recombination is a DNA rearrangement that performs a number of important biological functions, including integration of viral genomes into host chromosomes of bacteria, resolution of multimeric circular replicons such as plasmids and bacterial chromosomes to a monomeric state to ensure proper segregation upon cell division, amplification of plasmid copy number in yeast, and regulation of gene expression.
Cre recombinase is a member of the tyrosine recombinase family of enzymes that includes the bacteriophage λ integrase and the Flp recombinase from yeast. Cre mediates a site-specific recombination event at short DNA sequences called loxP. Because the reaction catalyzed by Cre can be performed both in the test tube and in virtually any living cell, this system has become widely used in the past decade as a tool in both in vitro and in vivo manipulations of DNA molecules. The serine integrases are a distinct family of site-specific recombinases with mechanisms of action that differ dramatically from the tyrosine recombinases. Several serine recombinases have recently emerged as powerful tools for execution of in vivo DNA integrations and show promise for future therapeutic applications.
To understand the detailed three-dimensional mechanisms of recombination and to provide a framework for engineering of specificity and function in site-specific recombinases, we have been studying Cre and a number of serine integrase systems. Our initial work focused on trapping intermediates of the Cre-loxP recombination reaction and using x-ray diffraction methods to determine their structures. These structural models have provided considerable insight into the three-dimensional nature of the recombination pathway. Our current work in the Cre system involves understanding the energetics of loxP-site synapsis and specificity within the Cre-DNA reaction complex. Current efforts in the serine recombinase systems are focused on obtaining three-dimensional structures of the catalytic domain and of integrase-DNA complexes. Our goal is to use this detailed structural and biochemical understanding of these systems to engineer a higher level of functionality, specificity, and control into the recombination pathways.
Poxvirus Nucleic Acid–Processing Enzymes
Poxviruses are a family of large DNA-containing viruses whose members are responsible for diseases such as smallpox and molluscum contagiosum. Although smallpox has been eradicated from the human population, it is presently feared as a possible agent of bioterrorism. Since viral replication occurs in the cytoplasm of infected cells, poxviruses code for their own DNA-processing enzymes. Among these are a number of potentially attractive drug targets, including the type IB topoisomerase, the DNA resolvase, and the DNA polymerase.
We have recently collaborated with Frederic Bushman (University of Pennsylvania) to determine the structure of the smallpox virus topoisomerase-DNA complex. Unlike the cellular topoisomerase, the viral topoisomerase is activated for catalysis only when bound to a specific DNA recognition sequence. Our goals are to establish the structural bases for specificity and activation of catalysis by the viral topoisomerase and to build a framework for the design of viral inhibitors. In principle, small molecules could be identified that act as viral topoisomerase poisons in a manner that is similar to the way anticancer agents such as camptothecin act on the human topoisomerase IB enzyme. We are also working to obtain similar structural frameworks for other poxvirus enzymes.
The SMN Complex
Spinal muscular atropy (SMA) is a common autosomal-recessive disease that is characterized by degeneration of motor neurons in the spinal cord. SMA is the leading hereditary cause of infant mortality, and mutations in the survival of motor neurons (SMN) gene have been shown to be responsible for the disease. The SMN protein (product of the SMN gene) is part of a large multiprotein complex in humans that is believed to be responsible for assembling the protein-RNA complexes (snRNPs) that remove intron sequences from mRNA. SMA may therefore be caused by a defect in the cell's ability to assemble its RNA-splicing machinery.
We are working to establish the structure of the SMN complex and to understand how this complex recognizes and assembles the protein and RNA components of the splicing factors. Using both the "divide and conquer" approach of studying individual proteins within the SMN complex and the more direct approach of studying larger subassemblies, we are attempting to establish a structural framework for understanding how this protein complex carries out its functions. Toward this goal, we have recently determined the high-resolution structure of the Gemin6/Gemin7 complex, in collaboration with the lab of Gideon Dreyfuss (HHMI, University of Pennsylvania).
In addition to increasing our understanding of the biological function of the SMN complex, we hope that an atomic resolution model revealing the consequences of commonly observed mutations in the SMN gene will provide insight into the development of therapies for the disease.
Last updated August 28, 2008
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