Characterizing the endoribonuclease activity of APE1.

Recent evidence shows that mRNA stability and turn-over is an integral control point in the regulation of gene expression. The stability of various mRNAs within a eukaryotic cell can differ and this results in a magnitude of difference in mRNA abundance. An enzyme known as APE1, apurinic/apyrimidinic DNA endonuclease 1, has recently been discovered to possess an endoribonuclease activity against c-myc messenger RNA (mRNA) in vitro. The identification of APE1 as an endoribonuclease warranted this research to further characterize this novel activity both in vitro and in vivo. Previous studies have discovered the residues constituting an active site for apurinic/aprymidinic [sic] DNA (AP-DNA) incision activity of APE1. Whether these residues are shared in the RNA-cleaving activity of APE1 was unknown. The first objective of this thesis was to assess the role of these amino acid residues in contributing to the endoribonuclease activity of APE1. Our results revealed that APE1 indeed shared these residues to cleave both RNA and AP-DNA. However, we also discovered certain differences in the activities of one mutant (D283N) in carrying out AP-DNA and RNA incisions. This suggested that the roles of active site residues in each reaction are not entirely identical. In addition, we have assessed the RNA-cleaving activities of APE1 variants identified in the human population. For a few variants, RNA-cleaving activities were severely reduced while its AP-DNA incision activities were functional. These results suggested a possible unrecognized link between the reductions in the RNA-cleaving activity of the variants and their reported association in certain diseases. The second objective of this thesis was to establish the RNA secondary structures and sequences that are preferentially cleaved by APE1. Our results revealed that APE1 has preference for cleaving the single stranded regions or weakly base paired regions of the RNA. Also, preferred sequences of cleavage were determined to be UA, UG, and CA dinucleotides. Prevalent A