Structural determinants for specific recognition by T4 endonuclease V.

The Journal of Biological Chemistry
A K McCulloughR S Lloyd

Abstract

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/apurinic (AP) lyase that is specific for UV light-induced cis-syn pyrimidine dimers. As a proposed transition state analog/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 assays and resulted in a single stable complex of reduced mobility and an apparent Kd of 17 nM. To assess the importance of the positive charge for specific binding, studies using other non-cleavable substrate analogs were performed. Wild type T4 endonuclease V shows an 8-fold decreased affinity for a tetrahydrofuran as compared with the pyrrolidine residue, demonstrating the significance of the positive charge for recognition. A 2-fold increase in binding affinity for a reduced AP site was observed. Similar assays using catalytically compromised mutants (E23Q and E23D) of endonuclease V demonstrate altered affinities for the pyrrolidine as well as tetrahydrofuran and reduced AP sites. This approach has provided insight into the structural mech...Continue Reading

Citations

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