Abasic sites are a form of DNA damage that arise through a number of pathways. These pathways occur during normal cellular processes, such as spontaneous base hydrolysis, reactions with oxygen radicals and other cellular metabolites, and abnormal events such as environmental insult. Unrepaired abasic sites can result in mutation and might be implicated in the development of cancer. The major human enzyme involved in abasic site recognition and removal is apurinic/apyrimidinic endonuclease (AP endo). The researchers would like to understand the basic mechanisms by which this enzyme functions as an endonuclease and as a participant in DNA repair. Towards that end, the researchers present a hypothesis describing how AP endo might recognize and interact with an abasic site from the viewpoint of the DNA containing the abasic site and from the viewpoint of the enzyme. The researchers also present preliminary data demonstrating that transient-state kinetics can be applied to the enzyme-DNA interaction to describe the minimum number of steps in the process of nicking DNA in a controlled fashion. These transient state studies were enabled by the discovery of an inhibitor so powerful that single turnover measurements were possible. This inhibitor might serve as the basis for drug development at a later time. The researchers then propose to use DNA binding and dissociation parameters to characterize how the enzyme interacts with substrate analogues: to map the domain structure of the enzyme in order to understand how different parts of the enzyme recognize and interact with the abasic site; and to determine what role specific enzymatic residues play in the enzyme-DNA interaction as defined by kinetic and structural measurements. These studies will contribute specific information on how AP endo recognizes and nicks an abasic site in preparation for damage repair.
