SPECIFIC BINDING OF A DESIGNED PYRROLIDINE ABASIC SITE ANALOG TO MULTIPLE DNA GLYCOSYLASES

In the base excision DNA repair pathway, DNA glycosylases recognize da maged bases in DNA and catalyze their excision through hydrolysis of t he N-glycosidic bond. Attempts to understand the structural basis for DNA damage recognition by DNA glycosylases have been hampered by the s hort-lived association of these enzymes with their DNA substrate. To o vercome this problem, we have employed an approach involving the desig n and synthesis of inhibitors that form stable complexes with DNA glyc osylases, which can then be studied biochemically and structurally. We have previously reported that double-stranded DNA containing a pyrrol idine abasic site analog (PYR) forms an extremely stable complex with the DNA glycosylase AlkA and potently inhibits the reaction catalyzed by the enzyme (Scharer, O. D., Ortholand, J.-Y., Ganesan, A., Ezaz-Nik pay, R., and Verdine, G. L. (1995) J. Am. Chem. Sec. 117, 6623-6624). Here we investigate the interaction of this inhibitor with a variety o f additional DNA glycosylases. With the exception of uracil DNA glycos ylase all the glycosylases tested bind specifically to PYR containing oligonucleotides. By comparing the interaction of DNA glycosylases wit h PYR and the structurally related tetrahydrofuran abasic site analog, we assess the importance of the positively charged ammonium group of the pyrrolidine in binding to the active site of these enzymes. Such a general inhibitor of DNA glycosyases provides a valuable tool to stud y stable complexes of these enzymes bound to substrate-like molecules.