Specific interactions of t4 endonuclease v with a proposed transition state analogue for dna glycosylases

DNA glycosylases catalyze the scission of the N-glycosyl bond linking either a damaged or mismatched base to the DNA sugar phosphate backbone. T4 endonuclease V is a glycosylase/AP lyase that is specific for UV light-induced cis-syn pyrimidine dimers. A unified catalytic mechanism for DNA glycosylases has been proposed in which the DNA glycosylases/AP lyases use a primary amino group as the attacking nucleophile resulting in an imino intermediate. As a proposed transition state analogue/inhibitor for glycosylases, a phosphoramidite derivative containing a pyrrolidine residue has been synthesized. The binding of endonuclease V to this duplex was analyzed by gel mobility shift assay and results in a single stable complex of reduced mobility. The apparent KD for the pyrrolidine-containing duplex is estimated at 1530 nM. Preliminary data using catalytically-compromised mutants (E23Q and E23D) of endonuclease V demonstrate altered affinities for ihe pyrrolidine as well as other non-cleavable site-specifically modified oligonucleotides, such as tetrahydrofuran, reduced AP site, and propanediol, as compared to the wild type endonuclease V. Initial fluorescence data on endonuclease V binding to these DNAs as well as the implications for the unified catalytic mechanism model will be discussed.