STRUCTURAL DETERMINANTS FOR SPECIFIC RECOGNITION BY T4 ENDONUCLEASE-V

DNA glycosylases catalyze the scission of the N-glycosyl bond linking either a damaged or mismatched base to the DNA sugar phosphate backbon e. T4 endonuclease V is a glycosylase/apurinic (AP) lyase that is spec ific for UV light-induced cis-syn pyrimidine dimers. As a proposed tra nsition state analog/inhibitor for glycosylases, a phosphoramidite der ivative containing a pyrrolidine residue has been synthesized. The bin ding of endonuclease V to this duplex was analyzed by gel mobility shi ft assays and resulted in a single stable complex of reduced mobility and an apparent K-d of 17 nM. To assess the importance of the positive charge for specific binding, studies using other non-cleavable substr ate analogs were performed. Wild type T4 endonuclease V shows an g-fol d decreased affinity for a tetrahydrofuran as compared with the pyrrol idine residue, demonstrating the significance of the positive charge f or recognition. A S-fold increase in binding affinity for a reduced AP site was observed. Similar assays using catalytically compromised mut ants (E23Q and E28D) of endonuclease V demonstrate altered affinities for the pyrrolidine as well as tetrahydrofuran and reduced AP sites. T his approach has provided insight into the structural mechanism by whi ch specific lesions are targeted by the protein as well as the structu ral determinants of the DNA required for specific recognition by T4 en donuclease V.