RECOGNITION OF SINGLE-STRANDED-DNA BY NUCLEASE P1 - HIGH-RESOLUTION CRYSTAL-STRUCTURES OF COMPLEXES WITH SUBSTRATE-ANALOGS

The reaction mechanism of nuclease P1 from Penicillium citrinum has be en investigated using single-stranded dithiophosphorylated di-, tetra- , and hexanucleotides as substrate analogs. The complexes crystallize in tetragonal and orthorhombic space groups and have been solved by mo lecular replacement. The high resolution structures give a clear pictu re of base recognition by P1 nuclease at its two nucleotide-binding si tes, especially the 1.8 Angstrom structure of a P1-tetranucleotide com plex which can be considered a P1-product complex. The observed bindin g modes are in agreement with a catalytic mechanism where the two clos ely spaced zinc ions activate the attacking water while the third, mor e exposed zinc ion stabilizes the leaving 03' oxyanion. Stacking as we ll as hydrogen bonding interactions with the base 5' to the cleaved ph osphodiester bond are important elements of substrate binding and reco gnition, Modelling of a productive P1-substrate complex based on the s olved structures suggests steric hindrance as the Likely reason for th e resistance of Rp-phosphorothioates and phosphorodithioates, Differen ces with the highly homologous nuclease S1 from Aspargillus oryzae are discussed. (C) 1998 Wiley-Liss, Inc.