Base excision repair (BER) is a critical pathway in repairing single base lesions caused by oxidation, deamination, and alkylation. NEIL3 is a DNA glycosylase which functions in initiating BER pathway by recognizing and removing damaged bases. Although there have been many studies reported on the function of NEIL3 in BER, the potential role of NEIL3 in the regulation of downstream BER players such as APE1 have not been dissected yet. In comparison to other Fpg/Nei glycosylase family members including NEIL1 and NEIL2, NEIL3 has two distinct zinc finger GRF motifs (Zf-GRF) in extreme C-terminus. In my thesis, two NEIL3 Zf-GRF motifs are my main focus due to their highly conserved residues across multiple vertebrates. Remarkably, my experimental observations reveal unexpected findings that two NEIL3 Zf-GRF motifs but not one Zf-GRF motif can interact and regulate APE1’s endonuclease activity. In particular, I have demonstrated (1) two NEIL3 Zf-GRF motifs associate with APE1 but not APE2, (2) NEIL3 Zf-GRF motifs bind to single-stranded DNA (ssDNA) in a sequence-independent manner, (3) two Zf-GRF motifs allow for binding to shorter ssDNA compared with one Zf-GRF motif, and (4) the Zf-GRF repeat within NEIL3 compromises APE1’s endonuclease activity on ssDNA. Taken together, my results suggest a distinct mechanism by which NEIL3 Zf-GRF repeat maintains genome stability of ssDNA via suppression of APE1 endonuclease activity-mediated DNA breaks.