Characterization of DNA and RNA end modifying enzymes, and, A triphosphate tunnel metalloenzyme

In all forms of life, DNA and RNA carry out the vital functions of transferring and storing genetic information. The essential nature of these molecules requires the presence of many enzymes and pathways to maintain and their integrity. Among these many enzymes are those involved in the repair, maintenance, and modification of DNA and RNA ends, including DNA and RNA ligases, polynucleotide kinase phosphatases, and RNA capping enzymes. All known ligases seal nicks with 3'-OH, 5'-PO4 termini within nucleic acids. Polynucleotide kinase phosphatase enzymes modify 3'-phosphate (or 2',3' cyclic phosphate) and 5'-OH termini to 3'-OH, 5'-PO4 ends suitable for ligation. RNA capping enzymes modify the 5' ends of RNA and triphosphatase members of this enzyme family serve as models for a larger class of triphosphatase tunnel metalloenzymes in general. This thesis discusses examples of each of these subgroups of end modifying and related enzymes.;DNA ligases are enzymes indispensible for DNA replication, recombination, and repair. The first step of the ligation reaction entails transfer of an AMP moiety to a lysine residue of the enzyme to form a covalent lysyl-AMP intermediate. Based on the substrate utilized DNA ligases are categorized as ATP or NAD+-dependent. Archaeal DNA ligases were thought to be strictly ATP-dependent. However recent reports suggest that dual specificity DNA ligases are encoded by members of the order thermococcales. It is plausible that the archaeal enzymes may represent an ancestral version of DNA ligases that utilize ADP to catalyze ligation. To test this hypothesis I cloned, purified and characterized a DNA ligase from the thermophilic archaeon Pyrococcus horikoshii.;Breaks in the phosphodiester bonds of DNA or RNA which results in 3'-PO 4 and 5'-OH termini must be healed before they can be sealed. Clostridium thermocellum Pnkp is the first RNA repair competent end-healing enzyme to be identified and characterized from a bacterium. The enzyme is unique with respect to its phosphatase domain. I characterized the phosphatase domain to gain insights on the mechanism of phosphoester hydrolysis and identify its substrate specificity. The findings are discussed in light of a possible physiological role of CthPnkp.;The Cet1-like RNA triphosphatases (from fungi, protozoa and viruses) and the CYTH family of enzymes comprise the triphosphate tunnel metalloenzyme superfamilly. The Cet1-like enzymes are involved in the first step of mRNA capping. The structurally and mechanistically related CYTH family of enzymes includes archaeal and bacterial proteins of unknown function. Most members are annotated as adenylate cyclases in the database. To test this prediction and gain an understanding of their physiological relevance, I cloned, purified and characterized a TTM from C. thermocellum.