title: Specific binding of a designed pyrrolidine abasic site analog to multiple DNA glycosylases.
abstract: In the base excision DNA repair pathway, DNA glycosylases recognize damaged bases in DNA and catalyze their excision through hydrolysis of the N-glycosidic bond. Attempts to understand the structural basis for DNA damage recognition by DNA glycosylases have been hampered by the short-lived association of these enzymes with their DNA substrates. To overcome this problem, we have employed an approach involving the design and synthesis of inhibitors that form stable complexes with DNA glycosylases, which can then be studied biochemically and structurally. We have previously reported that double-stranded DNA containing a pyrrolidine abasic site analog (PYR) forms an extremely stable complex with the DNA glycosylase AlkA and potently inhibits the reaction catalyzed by the enzyme (Schärer, O. D., Ortholand, J.-Y., Ganesan, A., Ezaz-Nikpay, K., and Verdine, G. L. (1995) J. Am. Chem. Soc. 117, 6623-6624). Here we investigate the interaction of this inhibitor with a variety of additional DNA glycosylases. With the exception of uracil DNA glycosylase all the glycosylases tested bind specifically to PYR-containing oligonucleotides. By comparing the interaction of DNA glycosylases with 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 study stable complexes of these enzymes bound to substrate-like molecules.
authors.affiliation: Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138, USA.
authors.forename: O D
authors.initials: OD
authors.lastname: Schärer
authors.forename: H M
authors.initials: HM
authors.lastname: Nash
authors.forename: J
authors.initials: J
authors.lastname: Jiricny
authors.forename: J
authors.initials: J
authors.lastname: Laval
authors.forename: G L
authors.initials: GL
authors.lastname: Verdine
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D004249:DNA Damage; D045647:DNA Glycosylases; D004260:DNA Repair; D005663:Furans; D007700:Kinetics; D009699:N-Glycosyl Hydrolases / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D011759:Pyrrolidines; D013379:Substrate Specificity
chemical_list: D005663:Furans; D009838:Oligodeoxyribonucleotides; D011759:Pyrrolidines; C018674:tetrahydrofuran; D004247:DNA; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; C076131:DNA-3-methyladenine glycosidase II