Cellular DNA is often damaged by oxidation, alkylation, and spontaneous deamination processes, as well as exposure to other exogenous agents. To preserve genome integrity, it is crucial for cells to recognize and repair DNA to its original encoding form. For damage occurring at a single base, DNA repair proceeds through the base excision repair (BER) pathway. BER is initiated by damage-specific glycosylases, such as Methyl Binding Domain IV (MBD4), which bind and excise specific types of damaged bases from DNA. The removal of these damaged bases results in the formation of an abasic or AP site, allowing the next enzyme in the pathway, Apurinic/Apyrimidinic Endonuclease 1 (APE1), to hydrolytically cleave the DNA backbone 5' to the lesion. Subsequent enzymes in the BER pathway then incorporate the correct nucleotide back into the DNA, excise the sugar fragment, and ligate the remaining nick, completing the repair process. It is important to maintain the repair pathways since mutations of BER enzymes can result in disease or cancer, and depletion of APE1 expression is correlated with embryonic lethality. This body of work predominantly focuses on the structural and biochemical characterization of the multi-functional APE1 protein. Using Nuclear Magnetic Resonance (NMR) spectroscopy, we assigned the backbone chemical shifts for APE1. This non-trivial assignment process required the development of a novel refolding protocol for the enzyme, which then enabled us to implement specialized TROSY NMR experiments. The assignments were used to identify the binding site for two redox (another APE1 function) inhibitors of APE1, one of which was surprisingly found to bind at the repair active site and inhibit its repair activity in contrast to previous proposals. We have also investigated the structure of APE1 using X-ray crystallography by determining a new crystallization condition for the enzyme. We crystallized wildtype and two natural variant APE1 proteins, Q51H and D148E, and found the variant proteins retain nearly identical structural integrity as the wildtype enzyme. The new crystallization condition will be useful for future structural studies of APE1, including crystallization with APE1 inhibitors. In addition to our studies of APE1, we have begun structural studies of MBD4, a BER glycosylase responsible for removing mispaired thymines from guanines within .a CpG context. We have obtained and solved the first crystal structure of the glycosylase domain of MBD4 bound to AP-DNA, enabling us to identify several residues likely involved in G:T recognition and catalysis. Altogether, the studies described herein provide new experimental strategies which can readily be used to study both enzymes, and our work provides significant insight into mechanisms and interactions involving APE1 and MBD4.