Abstract: Nucleases play an essential role in metabolism. They are involved in replication, recombination, restriction and repair1-3. Because of their participation in a large number of metabolic processes, the number of nucleases is vast and their distribution is ubiquitous. These enzymes are capable of promoting the hydrolysis of phosphodiester bond of nucleic acids with impressive rate enhancement1,4,5. In order to understand the molecular basis of the catalytic power of these enzymes, two approaches have been utilized. In the protein chemistry approach, structural and biochemical techniques were used to elucidate the structure and function of these nucleases. Since the number of nucleases is far too many to study, the work presented here focuses on one member of an endoribonuclease from an RNase III family in Saccharomyces cerevisiae, Rnt1p. Specifically, chapter four identifies a novel antideterminant within substrates of Rnt1p. This antideterminant has been shown to reduce the catalytic activity of the enzyme, perhaps by perturbing a structural element of Rnt1p. The identification of this antideterminant provides further evidence illustrating the complexity and intricacy of substrate recognition by S. cerevisiae, Rnt1p. Chapter five entails biochemical and genomic studies on the double-stranded RNA binding domain (dsRBD) of Rnt1p to probe for the molecular basis of substrate recognition. This chapter underscores a few important conserved residues and nucleotides on the dsRBD of Rnt1p and dsRNA substrate, respectively, that are essential for proper binding and cleavage of dsRNA by Rnt1p. Synthetic nucleases are good model systems for structural and mechanistic studies of natural nucleases. The design for effective chemical nucleases that have comparable catalytic power as natural nucleases has been a challenge and a struggle. Although there are a few chemical nucleases reported to exhibit relative rate increase compared to each other, the continuation in this synthetic approach is still highly encouraged because any information gained from these studies provides insight into new strategies to improve the design. Therefore chapter two of this dissertation presents a mechanistic study of a chemical nuclease, bisperoxovanadium (V) phenanthroline (bpVphen). The findings in this chapter finally identify the active species in the photochemical DNA strand scission by this complex.