Abasic sites are a form of DNA damage that arises through a number 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). We 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, we present a hypothesis describing how AP endo might recognize and interact with an abasic site from the viewpoint of the enzyme. In the past three years we have developed cutting-edge methodology to examine the kinetic behavior of the enzyme and prepared a number of site-directed mutant proteins to test that hypothesis. We have also completed the domain mapping of the enzyme in solution. In this application we report how our thoughts on the mechanism of action have evolved and describe how we propose to continue our efforts. We intend (1) to prepare particular site-directed mutant enzymes to further explore the mechanism of action, (2) to extend our quest for effective inhibitors of AP endo and (3) to examine the conformational changes that the enzyme probably undergoes in binding and nicking abasic site-containing DNA.
