The long term goals of this project are to develop a complete understanding of the biological roles and molecular mechanisms of the only known family of 3'-5' polymerases- enzymes that act in the opposite direction to all known DNA and RNA polymerases- in biology. The 3'-5' polymerase enzyme family contains tRNAHis guanylyltransferase (Thg1) proteins and Thg1-like proteins (TLPs). Thg1 proteins utilize the 3'-5' addition reaction to add a single required nucleotide to tRNAHis, which is an essential activity in many eukaryotes, including humans. On the other hand, although they share a related structure and basic catalytic mechanism, TLPs are biochemical and biologically distinct from Thg1, and the biological reactions that these enzymes catalyze are much less well-understood. At least one function of TLPs is to utilize Watson-Crick base pair dependent 3'-5' polymerase activity to add multiple nucleotides to repair the 5'-ends of tRNA in the mitochondria of many eukaryotic microbes. However, additional functions for these enzymes, including acting to repair or process other types of RNAs, are likely. Since RNA repair reactions are biologically important, and defects in these pathways can lead to negative effects on health, it is critical to fully understand the contributions of these unusual proteins to maintaining a healthy RNA pool. Interestingly, structures of several 3'-5' polymerases that are now available indicate that these enzymes share a distinct structural similarity and several aspects of their catalytic mechanism with canonical 5'-3' polymerases. Therefore, understanding the molecular basis for catalysis by 3'-5' polymerases is also important to understanding the distinctions between these two classes of nucleic acid synthesizing enzymes.
The specific aims of this proposal are to determine biological roles of 3'-5' polymerases in the slime mold, Dictystelium discoideum, as well as in some Archaea and S. cerevisiae. The molecular basis for substrate recognition, which is a key biological property that distinguishes Thg1 and TLPs, will also be investigated. This application proposes the use of kinetic, genetic, biochemical and structural techniques to investigate the molecular mechanisms and biological functions of both non-templated and templated 3'-5' addition reactions catalyzed by diverse 3'-5' polymerase family members. These results will provide insight into catalysis of a novel and apparently widespread, but largely unexplored, reaction in biology, and will enable further investigation into alternative functions for 3'-5' nucleotide addition in biological systems.
Investigation of the unusual 3'-5' nucleotide addition reactions catalyzed by Thg1 family members is of importance to human health, due to the absolute biological requirement for Thg1 activity for tRNAHis function in many eukaryotes including humans and the demonstrated link between Thg1 overexpression and diabetic nephropathy. Novel pathways of RNA editing and/or repair catalyzed by members of the 3'-5' polymerase family could contribute to human pathology if they are not able to contribute to maintaining a high quality RNA pool. A detailed understanding of the mechanism of Thg1 catalysis in diverse organisms may lead to identification of unique properties of Thg1 homologs from significant human pathogens, such as Plasmodium falciparum and Trichomonas vaginalis, which can be targeted for the development of new antiparasitic or antifungal agents.
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