Investigating the 3' RNA Phosphodiesterase and 3'-5' Exoribonuclease Activities of APE1.

Messenger RNA degradation is a major control point in determining the abundance of mRNA, and hence in the regulation of gene expression. Until recently, endoribonucleases, enzymes that cleave the phosphodiester bond within a polynucleotide chain, have been dismissed as the key enzymes responsible for mRNA turnover. The human apurinic/apyrimidinic endonuclease 1 (APE1) was recently identified as an endoribonuclease that is capable of cleaving c-myc mRNA in vitro and regulate c-myc mRNA level and half-life in cells. The ability to subject any target mRNA for degradation is a powerful therapeutic intervention, and the technologies currently in use have employed activities of endoribonucleases. Hence, in addition to increasing our basic knowledge on its role in the regulation of gene expression, a better understanding on the RNA-degrading enzyme such as APE1 has the potential in leading to the development of novel gene inactivation technology. The first objective of this thesis was to determine whether APE1 possesses 3'RNA phosphodiesterase and 3'-5' exoribonuclease activities. Using three difference RNA substrates, we confirmed that APE1 indeed has 3'RNA phosphodiesterase activity which is clearly absent in the common Ribonuclease A. We also found that Myricetin, a known inhibitor of AP-DNA activity, is capable of inhibiting 3'RNA phosphodiesterase activity of APE1. Our results also revealed that APE1 has weak to negligible 3'-5' exoribonuclease activity against unstructured poly-ribonucleotides. However, as with its known 3'DNA exonuclease activity, we found that APE1 has preferential in removing one nucleotide at 3' overhang of RNA. The second objective of this thesis was to determine the critical amino acids responsible for the 3' RNA phosphodiesterase activity of APE1. Our results revealed that APE1 uses several similar critical residues tin carrying out both its endoribonuclease and 3' APE1 uses several similar critical residues in carrying out