Investigating the Cellular Localization of APE1.

Apurinic/apyrimidinic endonuclease 1 (APE1) is a well-known DNA repair enzyme with multiple functions which include redox-activation, 3'-DNA phosphodiesterase, 3'-5' DNA exonuclease, and RNase H activities. Recently the novel function of APE1 in RNA metabolism has been demonstrated. APE1 was shown to possess the ability to cleaving single-stranded RNA as well as abasic RNA. It was also shown to physically interact with proteins involved in RNA metabolism. Previous studies have discovered the altered distribution and expression of APE1 in different cell types including cancer cells. The cytoplasmic distribution of APE1 has been shown to have correlation with aggressive carcinomas and poor prognoses for patients. This has led to an increasing number of studies on the undefined extra-nuclear roles of APE1 and APE1's potential implications in cancer development. In this thesis, I aimed (i) to confirm the importance of nuclear localization signal (NLS) of APE1 at the N-terminus, (ii) to discover the role of single nucleotide change of APE1 human population variants in its sub-cellular distribution, (iii) to assess the cellular localization of APE1 with processing bodies and stress granules under cellular stresses, and (iv) to initiate immune-fluorescence analysis of APE1 in breast cancer tissue. Our results revealed that APE1 indeed has an important NLS of 1-20 amino acids at the N-terminus and APE1 human population variants showed nuclear localizations identical to the wild-type APE1. There was no co-localization of APE1 with PBs and/or SBs implying that APE1 is unlikely to be involved in mRNA processing that is carried out in PBs and SGs. We observed redistribution of the ND20-APE1-GFP upon cellular stresses in HepG2 cells and this phenomenon has highlighted possible degradation of the cytoplasmic APE1 upon cellular stress. This finding is also consistent with previous discovery of the five critical lysine residues that are responsible for ubiquitination and APE1 degradation upon cellular stress. In summary, this