Determining the enzymatic mechanism for human AP endonuclease on a natural substrate

• Online Access: http://hdl.handle.net/2047/d20002579

Human AP endonuclease (Ape1) is a DNA repair protein responsible for the 5ʹ cleavage of abasic sites (AP sites), a major form of DNA damage. The multi-functional DNA repair protein also plays a role in cancer resistance to DNA damaging chemotherapeutics. This lab has shown that the enzyme cleaves a synthetic AP site by a one step mechanism. In an effort to develop a better understanding of Ape1 enzyme and provide relevant information for medicinal cancer research, I examined the enzymatic mechanism of Ape1 utilizing a natural AP site substrate, the reduced AP site. First, I evaluated the applicability of previous methodologies for the determination of the Ape1 cleavage mechanism on the reduced AP site substrate. Second, I attempted to resolve the mechanism for Ape1 cleavage of the reduced AP site employing electrospray ionization time of flight mass spectrometry (ESI-TOF-MS). The ESI-TOF-MS data validated that previous methodologies were applicable to a reduced AP site and support the use of this technology in future efforts to determine the Ape1 mechanism for cleavage on a reduced AP site substrate.