The Choreography of Base Excision Repair
The DNA in each cell of the human body is damaged more than 10,000 times every day. To avoid the mutagenic and cytotoxic effects that can be caused by the accumulation of DNA damage, human cells utilize DNA repair pathways such as base excision repair (BER). BER is carried out by a series of DNA repair proteins that function in a step-by-step process to identify, remove, and replace a damaged nucleotide. BER is critical to genome stability and cell survival, but unfortunately there are still aspects of BER that remain poorly understood. In this dissertation, we uncover new insight into the activities of two DNA repair proteins which act sequentially within the BER pathway: human apurinic/apyrimidinic endonuclease 1 (APE1) and DNA polymerase beta (Pol β). First, we report seven X-ray crystallographic structures that reveal how APE1 influences the conformations of a variety of different mismatched base pairs. We show that the conformation of DNA mismatches bound by APE1 differ from the conformation of similar mismatches bound by Pol β. These observed differences in base pairing hint at the difference in the forces that APE1 and Pol β impose on the DNA when bound. Next, we investigate the mechanism by which damaged DNA is transferred from APE1 to Pol β during BER. To do this, we constructed a single-molecule total internal reflection fluorescence (smTIRF) microscope and herein provide a detailed description of the design, assembly, and operation to assist other groups looking to employ smTIRF methods in their own lab. We utilize the smTIRF microscope to visualize, for the first time, the transfer of DNA between APE1 and Pol β in real-time. Our results indicate that, in the absence of other BER factors, transfer of the BER intermediate from APE1 to Pol β during BER is dependent on the dissociation kinetics of APE1 and the duration that Pol β remains bound near the APE1-5’ nick complex. Altogether, our work provides insight into the intermolecular interactions between APE1, Pol β, and DNA which facilitate the coordination of DNA repair.
Biochemistry, APE1, DNA Damage, DNA Repair, Polβ, single molecule, TIRF
Networked Digital Library of Theses and Dissertations